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Najjar FN, Williamson YM, Cooper HC, Barr JR, Williams TL. Optimization of Extraction Methods for the Quantification of Proteins in Mammalian Tissues. Anal Chem 2025; 97:10173-10179. [PMID: 40338194 DOI: 10.1021/acs.analchem.4c05751] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/09/2025]
Abstract
With the development of new vaccine technologies, such as mRNA vaccines, tissue studies are becoming increasingly important. Knowledge of the antigen expression amounts and where the antigen accumulates in the body is essential for designing safe and effective vaccines. Mammalian tissues present challenges in the detection and accurate quantification of target proteins because of their complexity and the lack of protocols that efficiently extract proteins with minimal sample loss. Here, we describe a protocol for the detection and accurate quantification of protein targets in commercially available snap-frozen lung, liver, kidney, and spleen of European domestic ferrets (Mustela putorius furo) by isotope dilution mass spectrometry (IDMS). Housekeeping proteins were chosen that range in abundance to account for different masses of tissue slices of the same organ. Target peptides used for IDMS quantification were conserved across several of the common animal model systems, including baby hamster kidney, mouse, and ferret. Hemagglutinin, the primary antigen of an influenza vaccine, was added at various concentrations to demonstrate the recovery of low-abundance proteins from the complex tissue homogenate. By using housekeeping proteins and a preparation protocol that minimizes sample loss, this study shows that IDMS can accurately quantify proteins in mammalian tissues with unmatched sensitivity and specificity.
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Affiliation(s)
- Fabio N Najjar
- Oak Ridge Institute for Science and Education, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, United States
| | - Yulanda M Williamson
- National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, United States
| | - Hans C Cooper
- National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, United States
| | - John R Barr
- National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, United States
| | - Tracie L Williams
- National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, United States
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2
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Rosin NL, Winstone TML, Kelley M, Biernaskie J, Dufour A, Orton DJ. Targeted proteomic approach for quantification of collagen type I and type III in formalin-fixed paraffin-embedded tissue. Sci Rep 2024; 14:17769. [PMID: 39090134 PMCID: PMC11294326 DOI: 10.1038/s41598-024-68377-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2024] [Accepted: 07/23/2024] [Indexed: 08/04/2024] Open
Abstract
Collagen is the most abundant protein in mammals and a major structural component of the extracellular matrix (ECM). Changes to ECM composition occur as a result of numerous physiological and pathophysiological causes, and a common means to evaluate these changes is the collagen 3 (Col3) to collagen 1 (Col1) ratio. Current methods to measure the Col3/1 ratio suffer from a lack of specificity and often under- or over-estimate collagen composition and quantity. This manuscript presents a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of Col3 and Col1 in FFPE tissues. Using surrogate peptides to generate calibration curves, Col3 and Col1 are readily quantified in FFPE tissue sections with high accuracy and precision. The method is applied to several tissue types from both human and reindeer sources, demonstrating its generalizability. In addition, the targeted LC-MS/MS method permits quantitation of the hydroxyprolinated form of Col3, which has significant implications for understanding not only the quantity of Col3 in tissue, but also understanding of the pathophysiology underlying many causes of ECM changes. This manuscript presents a straightforward, accurate, precise, and generalizable method for quantifying the Col3/1 ratio in a variety of tissue types and organisms.
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Affiliation(s)
- Nicole L Rosin
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
| | - Tara M L Winstone
- Department of Pathology and Laboratory Medicine, University of Calgary, 3535 Research Rd NW, Room 1E-415, Calgary, AB, T2I 2K8, Canada
| | - Margaret Kelley
- Department of Pathology and Laboratory Medicine, University of Calgary, 3535 Research Rd NW, Room 1E-415, Calgary, AB, T2I 2K8, Canada
| | - Jeff Biernaskie
- Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada
- Department of Surgery, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada
- Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada
- Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB, Canada
| | - Antoine Dufour
- Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada
- McCaig Institute for Bone and Joint Health, University of Calgary, Calgary, AB, Canada
| | - Dennis J Orton
- Department of Pathology and Laboratory Medicine, University of Calgary, 3535 Research Rd NW, Room 1E-415, Calgary, AB, T2I 2K8, Canada.
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3
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Joshi N, Garapati K, Ghose V, Kandasamy RK, Pandey A. Recent progress in mass spectrometry-based urinary proteomics. Clin Proteomics 2024; 21:14. [PMID: 38389064 PMCID: PMC10885485 DOI: 10.1186/s12014-024-09462-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Accepted: 02/12/2024] [Indexed: 02/24/2024] Open
Abstract
Serum or plasma is frequently utilized in biomedical research; however, its application is impeded by the requirement for invasive sample collection. The non-invasive nature of urine collection makes it an attractive alternative for disease characterization and biomarker discovery. Mass spectrometry-based protein profiling of urine has led to the discovery of several disease-associated biomarkers. Proteomic analysis of urine has not only been applied to disorders of the kidney and urinary bladder but also to conditions affecting distant organs because proteins excreted in the urine originate from multiple organs. This review provides a progress update on urinary proteomics carried out over the past decade. Studies summarized in this review have expanded the catalog of proteins detected in the urine in a variety of clinical conditions. The wide range of applications of urine analysis-from characterizing diseases to discovering predictive, diagnostic and prognostic markers-continues to drive investigations of the urinary proteome.
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Affiliation(s)
- Neha Joshi
- Manipal Academy of Higher Education (MAHE), Manipal, 576104, India
- Institute of Bioinformatics, International Technology Park, Bangalore, 560066, India
- Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA
| | - Kishore Garapati
- Manipal Academy of Higher Education (MAHE), Manipal, 576104, India
- Institute of Bioinformatics, International Technology Park, Bangalore, 560066, India
- Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA
| | - Vivek Ghose
- Manipal Academy of Higher Education (MAHE), Manipal, 576104, India
- Institute of Bioinformatics, International Technology Park, Bangalore, 560066, India
| | - Richard K Kandasamy
- Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA
- Department of Quantitative Health Sciences, Mayo Clinic, Rochester, MN, 55905, USA
- Center for Individualized Medicine, Mayo Clinic, Rochester, MN, 55905, USA
| | - Akhilesh Pandey
- Institute of Bioinformatics, International Technology Park, Bangalore, 560066, India.
- Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA.
- Department of Quantitative Health Sciences, Mayo Clinic, Rochester, MN, 55905, USA.
- Center for Individualized Medicine, Mayo Clinic, Rochester, MN, 55905, USA.
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Phipps WS, Kilgore MR, Kennedy JJ, Whiteaker JR, Hoofnagle AN, Paulovich AG. Clinical Proteomics for Solid Organ Tissues. Mol Cell Proteomics 2023; 22:100648. [PMID: 37730181 PMCID: PMC10692389 DOI: 10.1016/j.mcpro.2023.100648] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2022] [Revised: 09/07/2023] [Accepted: 09/12/2023] [Indexed: 09/22/2023] Open
Abstract
The evaluation of biopsied solid organ tissue has long relied on visual examination using a microscope. Immunohistochemistry is critical in this process, labeling and detecting cell lineage markers and therapeutic targets. However, while the practice of immunohistochemistry has reshaped diagnostic pathology and facilitated improvements in cancer treatment, it has also been subject to pervasive challenges with respect to standardization and reproducibility. Efforts are ongoing to improve immunohistochemistry, but for some applications, the benefit of such initiatives could be impeded by its reliance on monospecific antibody-protein reagents and limited multiplexing capacity. This perspective surveys the relevant challenges facing traditional immunohistochemistry and describes how mass spectrometry, particularly liquid chromatography-tandem mass spectrometry, could help alleviate problems. In particular, targeted mass spectrometry assays could facilitate measurements of individual proteins or analyte panels, using internal standards for more robust quantification and improved interlaboratory reproducibility. Meanwhile, untargeted mass spectrometry, showcased to date clinically in the form of amyloid typing, is inherently multiplexed, facilitating the detection and crude quantification of 100s to 1000s of proteins in a single analysis. Further, data-independent acquisition has yet to be applied in clinical practice, but offers particular strengths that could appeal to clinical users. Finally, we discuss the guidance that is needed to facilitate broader utilization in clinical environments and achieve standardization.
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Affiliation(s)
- William S Phipps
- Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, Washington, USA
| | - Mark R Kilgore
- Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, Washington, USA
| | - Jacob J Kennedy
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
| | - Jeffrey R Whiteaker
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
| | - Andrew N Hoofnagle
- Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, Washington, USA; Department of Medicine, University of Washington School of Medicine, Seattle, Washington, USA.
| | - Amanda G Paulovich
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA; Department of Medicine, University of Washington School of Medicine, Seattle, Washington, USA.
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Feldiorean A, Bena J, Nakashima MO, McShane AJ, Cotta CV. Formalin Fixation Followed by Paraffin Embedding Allows Long-Term Storage of Proteins for Liquid Chromatography-Tandem Mass Spectrometry Analysis. J Transl Med 2023; 103:100224. [PMID: 37517701 DOI: 10.1016/j.labinv.2023.100224] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2023] [Revised: 06/15/2023] [Accepted: 07/13/2023] [Indexed: 08/01/2023] Open
Abstract
In an anatomical pathology laboratory, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to characterize amyloid deposits identified in formalin-fixed paraffin-embedded tissue (FFPET). However, the development of additional tests is partially limited by the lack of information the passage of time has on the proteins in FFPET. To investigate the reliability of LC-MS/MS in the analysis of old FFPET specimens, 1 bone marrow aspirate clot was analyzed by LC-MS/MS yearly from 2014 to 2018, in 3 consecutive months. Peptide-spectrum match, number of peptides identified, and percentage of the proteins covered were the parameters collected for the hemoglobin subunits alpha (HbA), beta (HbB), delta (HbD), and gamma (HbG). These proteins are constant components of the peripheral blood and are present in high and low abundance, allowing the monitorization of the performance of the test across varying protein concentrations. The hemoglobin subunits were stable over the years studied; 71% to 74% of HbA, 77% to 80% of HbB, 69% to 77% of HbD, and 57% to 63% of HbG were covered, with no statistical difference between 2014 and 2018. The number of peptides identified was also constant, 11 to 13 for HbA, 13 to 15 for HbB, 11 to 14 for HbD, and 7 to 9 for HbG. Peptide spectrum match was only slightly more variable: 209 to 327 for HbA, 569 to 1052 for HbB, 286 to 533 HbD, and 142 to 292 for HbG. In conclusion, high abundance hemoglobins, HbA and HbB, and relatively low abundance ones, HbD and HbG, are preserved in FFPET and confidently identified by LC-MS/MS for at least 5 years.
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Affiliation(s)
| | - James Bena
- Department of Quantitative Health Sciences, Cleveland Clinic, Cleveland, Ohio
| | - Megan O Nakashima
- Department of Laboratory Medicine, RJ Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, Ohio
| | - Adam J McShane
- Department of Laboratory Medicine, RJ Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, Ohio
| | - Claudiu V Cotta
- Department of Laboratory Medicine, RJ Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, Ohio.
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Zeng W, Zhang W, Tse EHY, Liu J, Dong A, Lam KSW, Luan S, Kung WH, Chan TC, Cheung TH. Restoration of CPEB4 prevents muscle stem cell senescence during aging. Dev Cell 2023; 58:1383-1398.e6. [PMID: 37321216 DOI: 10.1016/j.devcel.2023.05.012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2021] [Revised: 03/24/2023] [Accepted: 05/19/2023] [Indexed: 06/17/2023]
Abstract
Age-associated impairments in adult stem cell functions correlate with a decline in somatic tissue regeneration capacity. However, the mechanisms underlying the molecular regulation of adult stem cell aging remain elusive. Here, we provide a proteomic analysis of physiologically aged murine muscle stem cells (MuSCs), illustrating a pre-senescent proteomic signature. During aging, the mitochondrial proteome and activity are impaired in MuSCs. In addition, the inhibition of mitochondrial function results in cellular senescence. We identified an RNA-binding protein, CPEB4, downregulated in various aged tissues, which is required for MuSC functions. CPEB4 regulates the mitochondrial proteome and activity through mitochondrial translational control. MuSCs devoid of CPEB4 induced cellular senescence. Importantly, restoring CPEB4 expression rescued impaired mitochondrial metabolism, improved geriatric MuSC functions, and prevented cellular senescence in various human cell lines. Our findings provide the basis for the possibility that CPEB4 regulates mitochondrial metabolism to govern cellular senescence, with an implication of therapeutic intervention for age-related senescence.
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Affiliation(s)
- Wenshu Zeng
- Division of Life Science, Center for Stem Cell Research, HKUST-Nan Fung Life Sciences Joint Laboratory, State Key Laboratory of Molecular Neuroscience, Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Wenxin Zhang
- Division of Life Science, Center for Stem Cell Research, HKUST-Nan Fung Life Sciences Joint Laboratory, State Key Laboratory of Molecular Neuroscience, Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Erin H Y Tse
- Division of Life Science, Center for Stem Cell Research, HKUST-Nan Fung Life Sciences Joint Laboratory, State Key Laboratory of Molecular Neuroscience, Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Jing Liu
- Division of Life Science, Center for Stem Cell Research, HKUST-Nan Fung Life Sciences Joint Laboratory, State Key Laboratory of Molecular Neuroscience, Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Anqi Dong
- Division of Life Science, Center for Stem Cell Research, HKUST-Nan Fung Life Sciences Joint Laboratory, State Key Laboratory of Molecular Neuroscience, Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Kim S W Lam
- Division of Life Science, Center for Stem Cell Research, HKUST-Nan Fung Life Sciences Joint Laboratory, State Key Laboratory of Molecular Neuroscience, Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Shaoyuan Luan
- Division of Life Science, Center for Stem Cell Research, HKUST-Nan Fung Life Sciences Joint Laboratory, State Key Laboratory of Molecular Neuroscience, Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Wai Hing Kung
- Division of Life Science, Center for Stem Cell Research, HKUST-Nan Fung Life Sciences Joint Laboratory, State Key Laboratory of Molecular Neuroscience, Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Tsz Ching Chan
- Division of Life Science, Center for Stem Cell Research, HKUST-Nan Fung Life Sciences Joint Laboratory, State Key Laboratory of Molecular Neuroscience, Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Tom H Cheung
- Division of Life Science, Center for Stem Cell Research, HKUST-Nan Fung Life Sciences Joint Laboratory, State Key Laboratory of Molecular Neuroscience, Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Hong Kong, China; Hong Kong Center for Neurodegenerative Diseases, Hong Kong, China; Guangdong Provincial Key Laboratory of Brain Science, Disease and Drug Development, Shenzhen-Hong Kong Institute of Brain Science, HKUST Shenzhen Research Institute, Shenzhen, China.
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7
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CPEB1 directs muscle stem cell activation by reprogramming the translational landscape. Nat Commun 2022; 13:947. [PMID: 35177647 PMCID: PMC8854658 DOI: 10.1038/s41467-022-28612-1] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Accepted: 01/14/2022] [Indexed: 11/08/2022] Open
Abstract
Skeletal muscle stem cells, also called Satellite Cells (SCs), are actively maintained in quiescence but can activate quickly upon extrinsic stimuli. However, the mechanisms of how quiescent SCs (QSCs) activate swiftly remain elusive. Here, using a whole mouse perfusion fixation approach to obtain bona fide QSCs, we identify massive proteomic changes during the quiescence-to-activation transition in pathways such as chromatin maintenance, metabolism, transcription, and translation. Discordant correlation of transcriptomic and proteomic changes reveals potential translational regulation upon SC activation. Importantly, we show Cytoplasmic Polyadenylation Element Binding protein 1 (CPEB1), post-transcriptionally affects protein translation during SC activation by binding to the 3' UTRs of different transcripts. We demonstrate phosphorylation-dependent CPEB1 promoted Myod1 protein synthesis by binding to the cytoplasmic polyadenylation elements (CPEs) within its 3' UTRs to regulate SC activation and muscle regeneration. Our study characterizes CPEB1 as a key regulator to reprogram the translational landscape directing SC activation and subsequent proliferation.
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8
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Laberiano-Fernández C, Hernández-Ruiz S, Rojas F, Parra ER. Best Practices for Technical Reproducibility Assessment of Multiplex Immunofluorescence. Front Mol Biosci 2021; 8:660202. [PMID: 34532339 PMCID: PMC8438151 DOI: 10.3389/fmolb.2021.660202] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Accepted: 08/11/2021] [Indexed: 11/22/2022] Open
Abstract
Multiplex immunofluorescence (mIF) tyramide signal amplification is a new and useful tool for the study of cancer that combines the staining of multiple markers in a single slide. Several technical requirements are important to performing high-quality staining and analysis and to obtaining high internal and external reproducibility of the results. This review manuscript aimed to describe the mIF panel workflow and discuss the challenges and solutions for ensuring that mIF panels have the highest reproducibility possible. Although this platform has shown high flexibility in cancer studies, it presents several challenges in pre-analytic, analytic, and post-analytic evaluation, as well as with external comparisons. Adequate antibody selection, antibody optimization and validation, panel design, staining optimization and validation, analysis strategies, and correct data generation are important for reproducibility and to minimize or identify possible issues during the mIF staining process that sometimes are not completely under our control, such as the tissue fixation process, storage, and cutting procedures.
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Affiliation(s)
- Caddie Laberiano-Fernández
- Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
| | - Sharia Hernández-Ruiz
- Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
| | - Frank Rojas
- Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
| | - Edwin Roger Parra
- Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States
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9
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Akturk G, Parra ER, Gjini E, Lako A, Lee JJ, Neuberg D, Zhang J, Yao S, Laface I, Rogic A, Chen PH, Sanchez-Espiridion B, Valle DMD, Moravec R, Kinders R, Hudgens C, Wu C, Wistuba II, Thurin M, Hewitt SM, Rodig S, Gnjatic S, Tetzlaff MT. Multiplex Tissue Imaging Harmonization: A Multicenter Experience from CIMAC-CIDC Immuno-Oncology Biomarkers Network. Clin Cancer Res 2021; 27:5072-5083. [PMID: 34253580 PMCID: PMC9777693 DOI: 10.1158/1078-0432.ccr-21-2051] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Revised: 07/05/2021] [Accepted: 07/06/2021] [Indexed: 01/07/2023]
Abstract
PURPOSE The Cancer Immune Monitoring and Analysis Centers - Cancer Immunologic Data Commons (CIMAC-CIDC) network supported by the NCI Cancer Moonshot initiative was established to provide correlative analyses for clinical trials in cancer immunotherapy, using state-of-the-art technology. Fundamental to this initiative is implementation of multiplex IHC assays to define the composition and distribution of immune infiltrates within tumors in the context of their potential role as biomarkers. A critical unanswered question involves the relative fidelity of such assays to reliably quantify tumor-associated immune cells across different platforms. EXPERIMENTAL DESIGN Three CIMAC sites compared across their laboratories: (i) image analysis algorithms, (ii) image acquisition platforms, (iii) multiplex staining protocols. Two distinct high-dimensional approaches were employed: multiplexed IHC consecutive staining on single slide (MICSSS) and multiplexed immunofluorescence (mIF). To eliminate variables potentially impacting assay performance, we completed a multistep harmonization process, first comparing assay performance using independent protocols followed by the integration of laboratory-specific protocols and finally, validating this harmonized approach in an independent set of tissues. RESULTS Data generated at the final validation step showed an intersite Spearman correlation coefficient (r) of ≥0.85 for each marker within and across tissue types, with an overall low average coefficient of variation ≤0.1. CONCLUSIONS Our results support interchangeability of protocols and platforms to deliver robust, and comparable data using similar tissue specimens and confirm that CIMAC-CIDC analyses may therefore be used with confidence for statistical associations with clinical outcomes largely independent of site, antibody selection, protocol, and platform across different sites.
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Affiliation(s)
- Guray Akturk
- Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York City, New York
| | - Edwin R Parra
- Translational Molecular Pathology-Dermatopathology Laboratory, The University of Texas MD Anderson Cancer Center, Houston, Texas
| | - Evisa Gjini
- Center for Immuno-Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Ana Lako
- Center for Immuno-Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - J Jack Lee
- Translational Molecular Pathology-Dermatopathology Laboratory, The University of Texas MD Anderson Cancer Center, Houston, Texas
| | | | - Jiexin Zhang
- Translational Molecular Pathology-Dermatopathology Laboratory, The University of Texas MD Anderson Cancer Center, Houston, Texas
| | - Shen Yao
- Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York City, New York
| | - Ilaria Laface
- Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York City, New York
| | - Anita Rogic
- Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York City, New York
| | | | - Beatriz Sanchez-Espiridion
- Translational Molecular Pathology-Dermatopathology Laboratory, The University of Texas MD Anderson Cancer Center, Houston, Texas
| | - Diane M Del Valle
- Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York City, New York
| | - Radim Moravec
- Kelly Services; Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH, Bethesda, Maryland
| | - Robert Kinders
- Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland
| | - Courtney Hudgens
- Translational Molecular Pathology-Dermatopathology Laboratory, The University of Texas MD Anderson Cancer Center, Houston, Texas
| | - Catherine Wu
- Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Ignacio I Wistuba
- Translational Molecular Pathology-Dermatopathology Laboratory, The University of Texas MD Anderson Cancer Center, Houston, Texas
| | - Magdalena Thurin
- Division of Cancer Treatment and Diagnosis, National Cancer Institute, NIH, Bethesda, Maryland
| | - Stephen M Hewitt
- Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland
| | - Scott Rodig
- Center for Immuno-Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
- Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Sacha Gnjatic
- Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York City, New York
| | - Michael T Tetzlaff
- Translational Molecular Pathology-Dermatopathology Laboratory, The University of Texas MD Anderson Cancer Center, Houston, Texas.
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Ahmed H, Ajat M, Mahmood RI, Mansor R, Razak ISA, Al-Obaidi JR, Razali N, Jaji AZ, Danmaigoro A, Bakar MZA. LC-MS/MS Proteomic Study of MCF-7 Cell Treated with Dox and Dox-Loaded Calcium Carbonate Nanoparticles Revealed Changes in Proteins Related to Glycolysis, Actin Signalling, and Energy Metabolism. BIOLOGY 2021; 10:biology10090909. [PMID: 34571787 PMCID: PMC8466983 DOI: 10.3390/biology10090909] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/08/2021] [Revised: 07/07/2021] [Accepted: 07/12/2021] [Indexed: 01/23/2023]
Abstract
Simple Summary This work revealed that DOX-Ar-CC-NPs have the ability to promote cell death in MCF-7 cells, showing high potency in drug delivery. DOX-Ar-CC-NPs prompts cell death of MCF-7 cancer cells in vivo. LC-MS/MS Proteomic experemnt showed alteration on the expression of proteins linked to actine signaling, carbohydrate metabolisim. Abstract One of the most prevalent death causes among women worldwide is breast cancer. This study aimed to characterise and differentiate the proteomics profiles of breast cancer cell lines treated with Doxorubicin (DOX) and Doxorubicin-CaCO3-nanoparticles (DOX-Ar-CC-NPs). This study determines the therapeutic potential of doxorubicin-loaded aragonite CaCO3 nanoparticles using a Liquid Chromatography/Mass Spectrometry analysis. In total, 334 proteins were expressed in DOX-Ar-CC-NPs treated cells, while DOX treatment expressed only 54 proteins. Out of the 334 proteins expressed in DOX-CC-NPs treated cells, only 36 proteins showed changes in abundance, while in DOX treated cells, only 7 out of 54 proteins were differentially expressed. Most of the 30 identified proteins that are differentially expressed in DOX-CC-NPs treated cells are key enzymes that have an important role in the metabolism of carbohydrates as well as energy, including: pyruvate kinase, ATP synthase, enolase, glyceraldehyde-3-phosphate dehydrogenase, mitochondrial ADP/ATP carrier, and trypsin. Other identified proteins are structural proteins which included; Keratin, α- and β-tubulin, actin, and actinin. Additionally, one of the heat shock proteins was identified, which is Hsp90; other proteins include Annexins and Human epididymis protein 4. While the proteins identified in DOX-treated cells were tubulin alpha-1B chain and a beta chain, actin cytoplasmic 1, annexin A2, IF rod domain-containing protein, and 78 kDa glucose-regulated protein. Bioinformatics analysis revealed the predicted canonical pathways linking the signalling of the actin cytoskeleton, ILK, VEGF, BAG2, integrin and paxillin, as well as glycolysis. This research indicates that proteomic analysis is an effective technique for proteins expression associated with chemotherapy drugs on cancer tumours; this method provides the opportunity to identify treatment targets for MCF-7 cancer cells, and a liquid chromatography-mass spectrometry (LC-MS/MS) system allowed the detection of a larger number of proteins than 2-DE gel analysis, as well as proteins with maximum pIs and high molecular weight.
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Affiliation(s)
- Hamidu Ahmed
- Natural Medicines and Products Research Laboratory (NaturMeds), Institute of Bioscience, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia;
- Department of Sciences and Engineering, Federal Polytechnic Mubi, P.M.B 35, Mubi 650221, Adamawa, Nigeria
| | - Mokrish Ajat
- Department of Veterinary Preclinical Science, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia; (M.A.); (R.M.); (I.S.A.R.); (A.Z.J.); (A.D.)
| | - Rana I. Mahmood
- Department of Biomedical Engineering, College of Engineering, Al-Nahrain University, Baghdad 64021, Iraq;
| | - Rozaihan Mansor
- Department of Veterinary Preclinical Science, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia; (M.A.); (R.M.); (I.S.A.R.); (A.Z.J.); (A.D.)
| | - Intan Shameha Abdul Razak
- Department of Veterinary Preclinical Science, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia; (M.A.); (R.M.); (I.S.A.R.); (A.Z.J.); (A.D.)
| | - Jameel R. Al-Obaidi
- Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, Tanjong Malim 35900, Perak, Malaysia
- Correspondence: (J.R.A.-O.); (M.Z.A.B.)
| | - Nurhanani Razali
- Membranology Unit, Okinawa Institute of Science and Technology Graduate University, 1919-1, Tancha, Onna-son, Kunigami-kun, Okinawa 904-0495, Japan;
| | - Alhaji Zubair Jaji
- Department of Veterinary Preclinical Science, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia; (M.A.); (R.M.); (I.S.A.R.); (A.Z.J.); (A.D.)
| | - Abubakar Danmaigoro
- Department of Veterinary Preclinical Science, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia; (M.A.); (R.M.); (I.S.A.R.); (A.Z.J.); (A.D.)
| | - Md Zuki Abu Bakar
- Natural Medicines and Products Research Laboratory (NaturMeds), Institute of Bioscience, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia;
- Correspondence: (J.R.A.-O.); (M.Z.A.B.)
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11
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Nakayasu ES, Gritsenko M, Piehowski PD, Gao Y, Orton DJ, Schepmoes AA, Fillmore TL, Frohnert BI, Rewers M, Krischer JP, Ansong C, Suchy-Dicey AM, Evans-Molina C, Qian WJ, Webb-Robertson BJM, Metz TO. Tutorial: best practices and considerations for mass-spectrometry-based protein biomarker discovery and validation. Nat Protoc 2021; 16:3737-3760. [PMID: 34244696 PMCID: PMC8830262 DOI: 10.1038/s41596-021-00566-6] [Citation(s) in RCA: 139] [Impact Index Per Article: 34.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Accepted: 04/26/2021] [Indexed: 02/06/2023]
Abstract
Mass-spectrometry-based proteomic analysis is a powerful approach for discovering new disease biomarkers. However, certain critical steps of study design such as cohort selection, evaluation of statistical power, sample blinding and randomization, and sample/data quality control are often neglected or underappreciated during experimental design and execution. This tutorial discusses important steps for designing and implementing a liquid-chromatography-mass-spectrometry-based biomarker discovery study. We describe the rationale, considerations and possible failures in each step of such studies, including experimental design, sample collection and processing, and data collection. We also provide guidance for major steps of data processing and final statistical analysis for meaningful biological interpretations along with highlights of several successful biomarker studies. The provided guidelines from study design to implementation to data interpretation serve as a reference for improving rigor and reproducibility of biomarker development studies.
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Affiliation(s)
- Ernesto S Nakayasu
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA.
| | - Marina Gritsenko
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA
| | - Paul D Piehowski
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA
| | - Yuqian Gao
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA
| | - Daniel J Orton
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA
| | - Athena A Schepmoes
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA
| | - Thomas L Fillmore
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA
| | - Brigitte I Frohnert
- Barbara Davis Center for Diabetes, School of Medicine, University of Colorado, Aurora, CO, USA
| | - Marian Rewers
- Barbara Davis Center for Diabetes, School of Medicine, University of Colorado, Aurora, CO, USA
| | - Jeffrey P Krischer
- Morsani College of Medicine, University of South Florida, Tampa, FL, USA
| | - Charles Ansong
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA
| | - Astrid M Suchy-Dicey
- Elson S. Floyd College of Medicine, Washington State University, Seattle, WA, USA
| | - Carmella Evans-Molina
- Center for Diabetes and Metabolic Diseases and the Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Wei-Jun Qian
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA
| | - Bobbie-Jo M Webb-Robertson
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA
- Department of Biostatistics and Informatics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
| | - Thomas O Metz
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA.
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12
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Proteomic comparison between different tissue preservation methods for identification of promising biomarkers of urothelial bladder cancer. Sci Rep 2021; 11:7595. [PMID: 33828141 PMCID: PMC8027873 DOI: 10.1038/s41598-021-87003-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2020] [Accepted: 03/22/2021] [Indexed: 11/08/2022] Open
Abstract
Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1-non-muscle invasive bladder cancer (NMIBC), and T2/T3-muscle invasive bladder cancer (MIBC)) which, upon sampling, were divided and preserved by FFPE and OCT. Samples were parallel processed from the two methods and proteins were analyzed with label-free quantitative MS. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Multivariate analysis indicates that the preservation method is the main source of variation, but also tumors of different stages could be differentiated. Proteins involved in mitochondrial function were overrepresented in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Concordant results for proteins such as HMGCS2 (uniquely quantified in Ta/T1 tumors), and LGALS1, ANXA5 and plastin (upregulated in T2/T3 tumors) were observed in both FFPE and OCT data, which supports the use of MS technology for biobank samples and encourages the further evaluation of these proteins as biomarkers.
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13
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Shidham VB. Cell-blocks and other ancillary studies (including molecular genetic tests and proteomics). Cytojournal 2021; 18:4. [PMID: 33880127 PMCID: PMC8053490 DOI: 10.25259/cytojournal_3_2021] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2021] [Accepted: 01/18/2021] [Indexed: 01/28/2023] Open
Abstract
Many types of elective ancillary tests may be required to support the cytopathologic interpretations. Most of these tests can be performed on cell-blocks of different cytology specimens. The cell-block sections can be used for almost any special stains including various histochemistry stains and for special stains for different microorganisms including fungi, Pneumocystis jirovecii (carinii), and various organisms including acid-fast organisms similar to the surgical biopsy specimens. Similarly, in addition to immunochemistry, different molecular tests can be performed on cell-blocks. Molecular tests broadly can be divided into two main types Molecular genetic tests and Proteomics.
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Affiliation(s)
- Vinod B Shidham
- Department of Pathology, Wayne State University School of Medicine, Karmanos Cancer Center, and Detroit Medical Center, Detroit, Michigan, United States
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14
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Chen Y, Sang C, Bian Z, Zhang Y, Jiang E, Zhou X, Chen T, Tang H, Wang C. The Scale, Collections, and Biospecimen Distribution of Grade A Tertiary Hospital Biobanks in China: A National Survey. Front Med (Lausanne) 2021; 7:560600. [PMID: 33537321 PMCID: PMC7848138 DOI: 10.3389/fmed.2020.560600] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2020] [Accepted: 12/21/2020] [Indexed: 11/13/2022] Open
Abstract
Chinese clinical biobanks were built rapidly in grade A tertiary hospitals. However, the general information of biorepositories in China remained largely unknown. The aim of this study was to investigate the size, collections, biospecimens distribution and other characteristics of Chinese biobanks in grade A tertiary hospitals. In 2018, we launched a national survey among biobank leaders to provide a comprehensive understanding of Chinese grade A tertiary hospital biobanks. A total of 70 biobank managers or directors completed an online questionnaire to collect information about the biorepositories. Nearly 20% of biobanks stored over one million specimens, while almost one-third of biobanks stored 50-200,000 specimens. In general, plasma and serum were the specimens most commonly stored. For the use of collections, biospecimens were most commonly applied by internal clinical departments. Further analyses revealed that the large-scale biobanks were characterized by earlier establishment, more types of specimens in storage and distribution compared with small-scale biobanks. Moreover, specimens in large-scale biobanks were more commonly used for basic research (62.86% vs. 34.29%, P = 0.017) and clinical research (57.14% vs. 28.57%, P = 0.016). Large-scale biobanks also had more opportunities to cooperate with domestic research institutes (34.29% vs. 5.71%, P = 0.003). Our survey revealed diversity in collections, distribution and utilization of biospecimens among Chinese grade A tertiary hospital biobanks. Although the biobanks had relatively large collections, the underutilization of stored biospecimens and lack of sharing could hamper clinical and biological research.
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Affiliation(s)
- Yuanyuan Chen
- Translational Medical Center for Stem Cell Therapy, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China.,Translational Medical Center for Stem Cell Therapy & Institute for Regenerative Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China
| | - Chao Sang
- Center for Translational Medicine, Shanghai Sixth People's Hospital, Shanghai, China
| | - Zhouliang Bian
- Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Yinan Zhang
- The Metabolic Diseases Biobank, Shanghai Key Laboratory of Diabetes, Shanghai Sixth People's Hospital, Shanghai, China
| | - Erpeng Jiang
- Translational Medical Center for Stem Cell Therapy, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China.,Translational Medical Center for Stem Cell Therapy & Institute for Regenerative Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China
| | | | - Tianlu Chen
- Center for Translational Medicine, Shanghai Sixth People's Hospital, Shanghai, China
| | - Hongming Tang
- Translational Medical Center for Stem Cell Therapy, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China.,Translational Medical Center for Stem Cell Therapy & Institute for Regenerative Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China
| | - Congrong Wang
- Department of Endocrinology, Shanghai Fourth People's Hospital Affiliated to Tongji University, Shanghai, China
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15
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Quantitative Proteomic Analysis Using Formalin-Fixed, Paraffin-Embedded (FFPE) Human Cardiac Tissue. Methods Mol Biol 2021; 2261:525-533. [PMID: 33421012 DOI: 10.1007/978-1-0716-1186-9_33] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Clinical tissue archives represent an invaluable source of biological information. Formalin-fixed, paraffin-embedded (FFPE) tissue can be used for retrospective investigation of biomarkers of diseases and prognosis.Recently, the number of studies using proteome profiling of samples from clinical archives has markedly increased. However, the application of conventional quantitative proteomics technologies remains a challenge mainly due to the harsh fixation process resulting in protein cross-linking and protein degradation. In the present chapter, we demonstrate a protocol for label-free proteomic analysis of FFPE tissue prepared from human cardiac autopsies. The data presented here highlight the applicability and suitability of FFPE heart tissue for understanding the molecular mechanism of cardiac injury using a proteomics approach.
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16
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Azimzadeh O, Azizova T, Merl-Pham J, Blutke A, Moseeva M, Zubkova O, Anastasov N, Feuchtinger A, Hauck SM, Atkinson MJ, Tapio S. Chronic Occupational Exposure to Ionizing Radiation Induces Alterations in the Structure and Metabolism of the Heart: A Proteomic Analysis of Human Formalin-Fixed Paraffin-Embedded (FFPE) Cardiac Tissue. Int J Mol Sci 2020; 21:ijms21186832. [PMID: 32957660 PMCID: PMC7555548 DOI: 10.3390/ijms21186832] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2020] [Revised: 09/14/2020] [Accepted: 09/14/2020] [Indexed: 12/23/2022] Open
Abstract
Epidemiological studies on workers employed at the Mayak plutonium enrichment plant have demonstrated an association between external gamma ray exposure and an elevated risk of ischemic heart disease (IHD). In a previous study using fresh-frozen post mortem samples of the cardiac left ventricle of Mayak workers and non-irradiated controls, we observed radiation-induced alterations in the heart proteome, mainly downregulation of mitochondrial and structural proteins. As the control group available at that time was younger than the irradiated group, we could not exclude age as a confounding factor. To address this issue, we have now expanded our study to investigate additional samples using archival formalin-fixed paraffin-embedded (FFPE) tissue. Importantly, the control group studied here is older than the occupationally exposed (>500 mGy) group. Label-free quantitative proteomics analysis showed that proteins involved in the lipid metabolism, sirtuin signaling, mitochondrial function, cytoskeletal organization, and antioxidant defense were the most affected. A histopathological analysis elucidated large foci of fibrotic tissue, myocardial lipomatosis and lymphocytic infiltrations in the irradiated samples. These data highlight the suitability of FFPE material for proteomics analysis. The study confirms the previous results emphasizing the role of adverse metabolic changes in the radiation-associated IHD. Most importantly, it excludes age at the time of death as a confounding factor.
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Affiliation(s)
- Omid Azimzadeh
- Helmholtz Zentrum München—German Research Centre for Environmental Health GmbH, Institute of Radiation Biology, 85764 Neuherberg, Germany; (N.A.); (M.J.A.); (S.T.)
- Correspondence: ; Tel.: +49-89-3187-3887
| | - Tamara Azizova
- Southern Urals Biophysics Institute (SUBI), Russian Federation, 456780 Ozyorsk, Russia; (T.A.); (M.M.); (O.Z.)
| | - Juliane Merl-Pham
- Helmholtz Zentrum München—German Research Centre for Environmental Health, Research Unit Protein Science, 80939 Munich, Germany; (J.M.-P.); (S.M.H.)
| | - Andreas Blutke
- Helmholtz Zentrum München—German Research Centre for Environmental Health GmbH, Research Unit Analytical Pathology, 85764 Neuherberg, Germany; (A.B.); (A.F.)
| | - Maria Moseeva
- Southern Urals Biophysics Institute (SUBI), Russian Federation, 456780 Ozyorsk, Russia; (T.A.); (M.M.); (O.Z.)
| | - Olga Zubkova
- Southern Urals Biophysics Institute (SUBI), Russian Federation, 456780 Ozyorsk, Russia; (T.A.); (M.M.); (O.Z.)
| | - Natasa Anastasov
- Helmholtz Zentrum München—German Research Centre for Environmental Health GmbH, Institute of Radiation Biology, 85764 Neuherberg, Germany; (N.A.); (M.J.A.); (S.T.)
| | - Annette Feuchtinger
- Helmholtz Zentrum München—German Research Centre for Environmental Health GmbH, Research Unit Analytical Pathology, 85764 Neuherberg, Germany; (A.B.); (A.F.)
| | - Stefanie M. Hauck
- Helmholtz Zentrum München—German Research Centre for Environmental Health, Research Unit Protein Science, 80939 Munich, Germany; (J.M.-P.); (S.M.H.)
| | - Michael J. Atkinson
- Helmholtz Zentrum München—German Research Centre for Environmental Health GmbH, Institute of Radiation Biology, 85764 Neuherberg, Germany; (N.A.); (M.J.A.); (S.T.)
- Chair of Radiation Biology, Technical University of Munich, 81675 Munich, Germany
| | - Soile Tapio
- Helmholtz Zentrum München—German Research Centre for Environmental Health GmbH, Institute of Radiation Biology, 85764 Neuherberg, Germany; (N.A.); (M.J.A.); (S.T.)
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17
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Do M, Kim H, Yeo I, Lee J, Park IA, Ryu HS, Kim Y. Clinical Application of Multiple Reaction Monitoring-Mass Spectrometry to Human Epidermal Growth Factor Receptor 2 Measurements as a Potential Diagnostic Tool for Breast Cancer Therapy. Clin Chem 2020; 66:1339-1348. [DOI: 10.1093/clinchem/hvaa178] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2020] [Accepted: 07/13/2020] [Indexed: 12/23/2022]
Abstract
Abstract
Background
Human epidermal growth factor receptor 2 (HER2) is often overexpressed in breast cancer and correlates with a worse prognosis. Thus, the accurate detection of HER2 is crucial for providing the appropriate measures for patients. However, the current techniques used to detect HER2 status, immunohistochemistry and fluorescence in situ hybridization (FISH), have limitations. Specifically, FISH, which is mandatory for arbitrating 2+ cases, is time-consuming and costly. To address this shortcoming, we established a multiple reaction monitoring-mass spectrometry (MRM-MS) assay that improves on existing methods for differentiating HER2 status.
Methods
We quantified HER2 expression levels in 210 breast cancer formalin-fixed paraffin-embedded (FFPE) tissue samples by MRM-MS. We aimed to improve the accuracy and precision of HER2 quantification by simplifying the sample preparation through predicting the number of FFPE slides required to ensure an adequate amount of protein and using the expression levels of an epithelial cell-specific protein as a normalization factor when measuring HER2 expression levels.
Results
To assess the correlation between MRM-MS and IHC/FISH data, HER2 quantitative data from MRM-MS were divided by the expression levels of junctional adhesion molecule A, an epithelial cell-specific protein, prior to statistical analysis. The normalized HER2 amounts distinguished between HER2 2+/FISH-negative and 2+/FISH-positive groups (AUROC = 0.908), which could not be differentiated by IHC. In addition, all HER2 status were discriminated by MRM-MS.
Conclusions
This MRM-MS assay yields more accurate HER2 expression levels relative to immunohistochemistry and should help to guide clinicians toward the proper treatment for breast cancer patients, based on their HER2 expression.
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Affiliation(s)
- Misol Do
- Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Hyunsoo Kim
- Department of Biomedical Engineering, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Injoon Yeo
- Department of Biomedical Engineering, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Jihyeon Lee
- Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - In Ae Park
- Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Han Suk Ryu
- Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Youngsoo Kim
- Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea
- Department of Biomedical Engineering, Seoul National University College of Medicine, Seoul, Republic of Korea
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18
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Byrling J, Kristl T, Hu D, Pla I, Sanchez A, Sasor A, Andersson R, Marko-Varga G, Andersson B. Mass spectrometry-based analysis of formalin-fixed, paraffin-embedded distal cholangiocarcinoma identifies stromal thrombospondin-2 as a potential prognostic marker. J Transl Med 2020; 18:343. [PMID: 32887625 PMCID: PMC7487897 DOI: 10.1186/s12967-020-02498-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2020] [Accepted: 08/21/2020] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND Distal cholangiocarcinoma is an aggressive malignancy with a dismal prognosis. Diagnostic and prognostic biomarkers for distal cholangiocarcinoma are lacking. The aim of the present study was to identify differentially expressed proteins between distal cholangiocarcinoma and normal bile duct samples. METHODS A workflow utilizing discovery mass spectrometry and verification by parallel reaction monitoring was used to analyze surgically resected formalin-fixed, paraffin-embedded samples from distal cholangiocarcinoma patients and normal bile duct samples. Bioinformatic analysis was used for functional annotation and pathway analysis. Immunohistochemistry was performed to validate the expression of thrombospondin-2 and investigate its association with survival. RESULTS In the discovery study, a total of 3057 proteins were identified. Eighty-seven proteins were found to be differentially expressed (q < 0.05 and fold change ≥ 2 or ≤ 0.5); 31 proteins were upregulated and 56 were downregulated in the distal cholangiocarcinoma samples compared to controls. Bioinformatic analysis revealed an abundance of differentially expressed proteins associated with the tumor reactive stroma. Parallel reaction monitoring verified 28 proteins as upregulated and 18 as downregulated in distal cholangiocarcinoma samples compared to controls. Immunohistochemical validation revealed thrombospondin-2 to be upregulated in distal cholangiocarcinoma epithelial and stromal compartments. In paired lymph node metastases samples, thrombospondin-2 expression was significantly lower; however, stromal thrombospondin-2 expression was still frequent (72%). Stromal thrombospondin-2 was an independent predictor of poor disease-free survival (HR 3.95, 95% CI 1.09-14.3; P = 0.037). CONCLUSION Several proteins without prior association with distal cholangiocarcinoma biology were identified and verified as differentially expressed between distal cholangiocarcinoma and normal bile duct samples. These proteins can be further evaluated to elucidate their biomarker potential and role in distal cholangiocarcinoma carcinogenesis. Stromal thrombospondin-2 is a potential prognostic marker in distal cholangiocarcinoma.
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Affiliation(s)
- Johannes Byrling
- Department of Clinical Sciences Lund, Surgery, Lund University, and Skåne University Hospital, Lund, Sweden
| | - Theresa Kristl
- Department of Biomedical Engineering, Clinical Protein Science and Imaging, Lund University, Lund, Sweden
| | - Dingyuan Hu
- Department of Clinical Sciences Lund, Surgery, Lund University, and Skåne University Hospital, Lund, Sweden
| | - Indira Pla
- Department of Biomedical Engineering, Clinical Protein Science and Imaging, Lund University, Lund, Sweden
| | - Aniel Sanchez
- Department of Biomedical Engineering, Clinical Protein Science and Imaging, Lund University, Lund, Sweden
| | - Agata Sasor
- Department of Clinical Sciences Lund, Pathology, Lund University, and Skåne University Hospital, Lund, Sweden
| | - Roland Andersson
- Department of Clinical Sciences Lund, Surgery, Lund University, and Skåne University Hospital, Lund, Sweden
| | - György Marko-Varga
- Department of Biomedical Engineering, Clinical Protein Science and Imaging, Lund University, Lund, Sweden
| | - Bodil Andersson
- Department of Clinical Sciences Lund, Surgery, Lund University, and Skåne University Hospital, Lund, Sweden.
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Establishment and validation of highly accurate formalin-fixed paraffin-embedded quantitative proteomics by heat-compatible pressure cycling technology using phase-transfer surfactant and SWATH-MS. Sci Rep 2020; 10:11271. [PMID: 32647189 PMCID: PMC7347883 DOI: 10.1038/s41598-020-68245-2] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2020] [Accepted: 06/22/2020] [Indexed: 11/08/2022] Open
Abstract
The purpose of this study was to establish a quantitative proteomic method able to accurately quantify pathological changes in the protein expression levels of not only non-membrane proteins, but also membrane proteins, using formalin-fixed paraffin-embedded (FFPE) samples. Protein extraction from FFPE sections of mouse liver was increased 3.33-fold by pressure cycling technology (PCT) and reached the same level as protein extraction from frozen sections. After PCT-assisted processing of FFPE liver samples followed by SWATH-MS-based comprehensive quantification, the peak areas of 88.4% of peptides agreed with those from matched fresh samples within a 1.5-fold range. For membrane proteins, this percentage was remarkably increased from 49.1 to 93.8% by PCT. Compared to the conventional method using urea buffer, the present method using phase-transfer surfactant (PTS) buffer at 95 °C showed better agreement of peptide peak areas between FFPE and fresh samples. When our method using PCT and PTS buffer at 95 °C was applied to a bile duct ligation (BDL) disease model, the BDL/control expression ratios for 80.0% of peptides agreed within a 1.2-fold range between FFPE and fresh samples. This heat-compatible FFPE-PCT-SWATH proteomics technology using PTS is suitable for quantitative studies of pathological molecular mechanisms and biomarker discovery utilizing widely available FFPE samples.
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20
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The Determination of Immunomodulation and Its Impact on Survival of Rectal Cancer Patients Depends on the Area Comprising a Tissue Microarray. Cancers (Basel) 2020; 12:cancers12030563. [PMID: 32121328 PMCID: PMC7139832 DOI: 10.3390/cancers12030563] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2019] [Revised: 02/02/2020] [Accepted: 02/17/2020] [Indexed: 12/26/2022] Open
Abstract
BACKGROUND T cell density in colorectal cancer (CRC) has proven to be of high prognostic importance. Here, we evaluated the influence of a hyperfractionated preoperative short-term radiation protocol (25 Gy) on immune cell density in tumor samples of rectal cancer (RC) patients and on patient survival. In addition, we assessed spatial tumor heterogeneity by comparison of analogue T cell quantification on full tissue sections with digital T cell quantification on a virtually established tissue microarray (TMA). METHODS A total of 75 RC patients (60 irradiated, 15 treatment-naïve) were defined for retrospective analysis. RC samples were processed for immunohistochemistry (CD3, CD8, PD-1, PD-L1). Analogue (score 0-3) as well as digital quantification (TMA: 2 cores vs. 6 cores, mean T cell count) of marker expression in 2 areas (central tumor, CT; invasive margin, IM) was performed. Survival was estimated on the basis of analogue as well as digital marker densities calculated from 2 cores (Immunoscore: CD3/CD8 ratio) and 6 cores per tumor area. RESULTS Irradiated RC samples showed a significant decrease in CD3 and CD8 positive T cells, independent of quantification mode. T cell densities of 6 virtual cores approximated to T cell densities of full tissue sections, independent of individual core density or location. Survival analysis based on full tissue section quantification demonstrated that CD3 and CD8 positive T cells as well as PD-1 positive tumor infiltrating leucocytes (TILs) in the CT and the IM had a significant impact on disease-free survival (DFS) as well as overall survival (OS). In addition, CD3 and CD8 positive T cells as well as PD-1 positive TILs in the IM proved as independent prognostic factors for DFS and OS; in the CT, PD-1 positive TILs predicted DFS and CD3 and CD8 positive T cells as well as PD-1 positive TILs predicted OS. Survival analysis based on virtual TMA showed no impact on DFS or OS. CONCLUSION Spatial tumor heterogeneity might result in inadequate quantification of immune marker expression; however, if using a TMA, 6 cores per tumor area and patient sample represent comparable amounts of T cell densities to those quantified on full tissue sections. Consistently, the tissue area used for immune marker quantification represents a crucial factor for the evaluation of prognostic and predictive biomarker potential.
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21
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Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies. Cancers (Basel) 2020; 12:cancers12020255. [PMID: 31972974 PMCID: PMC7072187 DOI: 10.3390/cancers12020255] [Citation(s) in RCA: 62] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2019] [Revised: 01/13/2020] [Accepted: 01/15/2020] [Indexed: 12/22/2022] Open
Abstract
In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.
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22
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Abstract
Multiplexed imaging platforms to simultaneously detect multiple epitopes in the same tissue section emerged in the last years as very powerful tools to study tumor immune contexture. These revolutionary technologies are providing a deep methodology for tumor evaluation in formalin-fixed and paraffin-embedded (FFPE) to improve the understanding of tumor microenvironment, new targets for treatment, prognostic and predictive biomarkers, and translational studies. Multiplexed imaging platforms allow for the identification of several antigens simultaneously from a single tissue section, core needle biopsies, and tissue microarrays. In recent years, multiplexed imaging has improved the abilities to characterize the different types of cell populations in malignant and non-malignant tissues, and their spatial distribution in relationship to clinical outcomes. Multiplexed technologies associated with digital image analysis software offer a high-quality throughput assay to study cancer specimens at multiple time points before, during and after treatment. The aim of this chapter is to provide a review of multiplexed imaging covering its fundamentals, advantages, disadvantages, and material and methods for staining applied to FFPE tumor tissues and focusing on the use of multiplex immunofluorescence with tyramine signal amplification staining for immune profiling and translational research.
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23
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Condina MR, Mittal P, Briggs MT, Oehler MK, Klingler-Hoffmann M, Hoffmann P. Egg White as a Quality Control in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI). Anal Chem 2019; 91:14846-14853. [PMID: 31660720 DOI: 10.1021/acs.analchem.9b03091] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The strength of MALDI-MSI is to analyze and visualize spatial intensities of molecular features from an intact tissue. The distribution of the intensities can then be visualized within a single tissue section or compared in between sections, acquired consecutively. This method can be reliably used to reveal physiological structures and has the potential to identify molecular details, which correlate with biological outcomes. MALDI-MSI implementation in clinical laboratories requires the ability to ensure method quality and validation to meet diagnostic expectations. To be able to get consistent qualitative and quantitative results, standardized sample preparation and data acquisition are of highest priority. We have previously shown that the deposition of internal standards onto the tissue section during sample preparation can be used to improve the mass accuracy of monitored m/z features across the sample. Here, we present the use of external and internal controls for the quality check of sample preparation and data acquisition, which is particularly relevant when either many spectra are acquired during a single MALDI-MSI experiment or data from independent experiments are processed together. To monitor detector performance and sample preparation, we use egg white as an external control for peptide and N-glycan MALDI-MSI throughout the experiment. We have also identified endogenous peptides from cytoskeletal proteins, which can be reliably monitored in gynecological tissue samples. Lastly, we summarize our standard quality control workflow designed to produce reliable and comparable MALDI-MSI data from single sections and tissue microarrays (TMAs).
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Affiliation(s)
- Mark R Condina
- Future Industries Institute , The University of South Australia , Adelaide , South Australia 5095 , Australia
| | - Parul Mittal
- Adelaide Proteomics Centre , The University of Adelaide , Adelaide , South Australia 5005 , Austrailia
| | - Matthew T Briggs
- Future Industries Institute , The University of South Australia , Adelaide , South Australia 5095 , Australia
| | - Martin K Oehler
- Discipline of Obstetrics and Gynaecology, Adelaide Medical School, Robinson Research Institute , The University of Adelaide , Adelaide , South Australia 5000 , Australia.,Department of Gynaecological Oncology , Royal Adelaide Hospital , Adelaide , South Australia 5005 , Australia
| | - Manuela Klingler-Hoffmann
- Future Industries Institute , The University of South Australia , Adelaide , South Australia 5095 , Australia
| | - Peter Hoffmann
- Future Industries Institute , The University of South Australia , Adelaide , South Australia 5095 , Australia
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24
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Jorge S, Capelo JL, LaFramboise W, Dhir R, Lodeiro C, Santos HM. Development of a Robust Ultrasonic-Based Sample Treatment To Unravel the Proteome of OCT-Embedded Solid Tumor Biopsies. J Proteome Res 2019; 18:2979-2986. [PMID: 31173681 DOI: 10.1021/acs.jproteome.9b00248] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
An effective three-step proteomics workflow is proposed to overcome the pitfalls caused by polymers present in optimum cutting temperature (OCT)-embedded tissue during its preparation for mass spectrometry analysis. First, the OCT-embedded tissue biopsies are cleaned using ethanol and water in a sequential series of ultrasonic washes in an ultrasound bath (35 kHz ultrasonic frequency, 100% ultrasonic amplitude, 2 min of ultrasonic duty time). Second, a fast ultrasonic-assisted extraction of proteins is done using an ultrasonic probe (30 kHz ultrasonic frequency, 50% ultrasonic amplitude, 2 min of ultrasonic duty time, 1 mm diameter tip). Third, a rapid ultrasonic digestion of complex proteomes is performed using a microplate horn assembly device (20 kHz ultrasonic frequency, 25% ultrasonic amplitude, 4 min of ultrasonic duty time). As a proof of concept, the new workflow was applied to human normal and tumor kidney biopsies including chromophobe renal cell carcinomas (chRCCs) and renal oncocytomas (ROs). A successful cluster of proteomics profiles was obtained comprising 511 and 172 unique proteins found in chRCC and RO samples, respectively. The new method provides high sample throughput and comprehensive protein recovery from OCT samples.
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Affiliation(s)
- Susana Jorge
- BIOSCOPE Research Group, LAQV, REQUIMTE, Department of Chemistry, Faculdade de Ciências e Tecnologia , Universidade NOVA de Lisboa , 2829-516 Caparica , Portugal.,PROTEOMASS Scientific Society , Madan Park, Rua dos Inventores , 2825-152 Caparica , Portugal
| | - José L Capelo
- BIOSCOPE Research Group, LAQV, REQUIMTE, Department of Chemistry, Faculdade de Ciências e Tecnologia , Universidade NOVA de Lisboa , 2829-516 Caparica , Portugal.,PROTEOMASS Scientific Society , Madan Park, Rua dos Inventores , 2825-152 Caparica , Portugal
| | - William LaFramboise
- Department of Pathology , University of Pittsburgh Medical Center , Pittsburgh , Pennsylvania 15261 , United States
| | - Rajiv Dhir
- Department of Pathology , University of Pittsburgh Medical Center , Pittsburgh , Pennsylvania 15261 , United States
| | - Carlos Lodeiro
- BIOSCOPE Research Group, LAQV, REQUIMTE, Department of Chemistry, Faculdade de Ciências e Tecnologia , Universidade NOVA de Lisboa , 2829-516 Caparica , Portugal.,PROTEOMASS Scientific Society , Madan Park, Rua dos Inventores , 2825-152 Caparica , Portugal
| | - Hugo M Santos
- BIOSCOPE Research Group, LAQV, REQUIMTE, Department of Chemistry, Faculdade de Ciências e Tecnologia , Universidade NOVA de Lisboa , 2829-516 Caparica , Portugal.,PROTEOMASS Scientific Society , Madan Park, Rua dos Inventores , 2825-152 Caparica , Portugal
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25
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Ducret A, James I, Wilson S, Feilke M, Tebbe A, Dybowski N, Elschenbroich S, Klammer M, Blackler A, Liao WL, Tian Y, Friess T, Bossenmaier B, Dietmann G, Schaab C, Hembrough T, Ceppi M. Translation and evaluation of a pre-clinical 5-protein response prediction signature in a breast cancer phase Ib clinical trial. PLoS One 2019; 14:e0213892. [PMID: 30897176 PMCID: PMC6428264 DOI: 10.1371/journal.pone.0213892] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2018] [Accepted: 03/05/2019] [Indexed: 12/21/2022] Open
Abstract
Human protein biomarker discovery relies heavily on pre-clinical models, in particular established cell lines and patient-derived xenografts, but confirmation studies in primary tissue are essential to demonstrate clinical relevance. We describe in this study the process that was followed to clinically translate a 5-protein response signature predictive for the activity of an anti-HER3 monoclonal antibody (lumretuzumab) originally measured in fresh frozen xenograft tissue. We detail the development, qualification, and validation of the multiplexed targeted mass spectrometry assay used to assess the signature performance in formalin-fixed, paraffin-embedded human clinical samples collected in a phase Ib trial designed to evaluate lumretuzumab in patients with metastatic breast cancer. We believe that the strategy delineated here provides a path forward to avoid the time- and cost-consuming step of having to develop immunological reagents against unproven targets. We expect that mass spectrometry-based platforms may become part of a rational process to rapidly test and qualify large number of candidate biomarkers to identify the few that stand a chance for further development and validation.
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Affiliation(s)
- Axel Ducret
- Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland
| | - Ian James
- A4P Consulting Ltd, Sandwich, United Kingdom
| | - Sabine Wilson
- Roche Pharmaceutical Research and Early Development, Roche Innovation Center Munich, Penzberg, Germany
| | - Martina Feilke
- Roche Pharmaceutical Research and Early Development, Roche Innovation Center Munich, Penzberg, Germany
| | | | | | | | | | - Adele Blackler
- Oncoplex Diagnostics, Rockville, MD, United States of America
| | - Wei-Li Liao
- Oncoplex Diagnostics, Rockville, MD, United States of America
| | - Yuan Tian
- Oncoplex Diagnostics, Rockville, MD, United States of America
| | - Thomas Friess
- Roche Pharmaceutical Research and Early Development, Roche Innovation Center Munich, Penzberg, Germany
| | - Birgit Bossenmaier
- Roche Pharmaceutical Research and Early Development, Roche Innovation Center Munich, Penzberg, Germany
| | - Gabriele Dietmann
- Roche Pharmaceutical Research and Early Development, Roche Innovation Center Munich, Penzberg, Germany
| | | | - Todd Hembrough
- Oncoplex Diagnostics, Rockville, MD, United States of America
| | - Maurizio Ceppi
- Roche Pharmaceutical Research and Early Development, Roche Innovation Center Munich, Penzberg, Germany
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26
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Spodzieja M, Rodziewicz-Motowidło S, Szymanska A. Hyphenated Mass Spectrometry Techniques in the Diagnosis of Amyloidosis. Curr Med Chem 2019; 26:104-120. [DOI: 10.2174/0929867324666171003113019] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2016] [Revised: 07/25/2016] [Accepted: 09/01/2016] [Indexed: 12/18/2022]
Abstract
Amyloidoses are a group of diseases caused by the extracellular deposition of proteins forming amyloid fibrils. The amyloidosis is classified according to the main protein or peptide that constitutes the amyloid fibrils. The most effective methods for the diagnosis of amyloidosis are based on mass spectrometry. Mass spectrometry enables confirmation of the identity of the protein precursor of amyloid fibrils in biological samples with very high sensitivity and specificity, which is crucial for proper amyloid typing. Due to the fact that biological samples are very complex, mass spectrometry is usually connected with techniques such as liquid chromatography or capillary electrophoresis, which enable the separation of proteins before MS analysis. Therefore mass spectrometry constitutes an important part of the so called “hyphenated techniques” combining, preferentially in-line, different analytical methods to provide comprehensive information about the studied problem. Hyphenated methods are very useful in the discovery of biomarkers in different types of amyloidosis. In systemic forms of amyloidosis, the analysis of aggregated proteins is usually performed based on the tissues obtained during a biopsy of an affected organ or a subcutaneous adipose tissue. In some cases, when the diagnostic biopsy is not possible due to the fact that amyloid fibrils are formed in organs like the brain (Alzheimer’s disease), the study of biomarkers presented in body fluids can be carried out. Currently, large-scale studies are performed to find and validate more effective biomarkers, which can be used in diagnostic procedures. We would like to present the methods connected with mass spectrometry which are used in the diagnosis of amyloidosis based on the analysis of proteins occurring in tissues, blood and cerebrospinal fluid.
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Affiliation(s)
- Marta Spodzieja
- Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland
| | - Sylwia Rodziewicz-Motowidło
- Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland
| | - Aneta Szymanska
- Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland
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27
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State-of-the-Art of Profiling Immune Contexture in the Era of Multiplexed Staining and Digital Analysis to Study Paraffin Tumor Tissues. Cancers (Basel) 2019; 11:cancers11020247. [PMID: 30791580 PMCID: PMC6406364 DOI: 10.3390/cancers11020247] [Citation(s) in RCA: 82] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2019] [Revised: 02/12/2019] [Accepted: 02/14/2019] [Indexed: 02/07/2023] Open
Abstract
Multiplexed platforms for multiple epitope detection have emerged in the last years as very powerful tools to study tumor tissues. These revolutionary technologies provide important visual techniques for tumor examination in formalin-fixed paraffin-embedded specimens to improve the understanding of the tumor microenvironment, promote new treatment discoveries, aid in cancer prevention, as well as allowing translational studies to be carried out. The aim of this review is to highlight the more recent methodologies that use multiplexed staining to study simultaneous protein identification in formalin-fixed paraffin-embedded tumor tissues for immune profiling, clinical research, and potential translational analysis. New multiplexed methodologies, which permit the identification of several proteins at the same time in one single tissue section, have been developed in recent years with the ability to study different cell populations, cells by cells, and their spatial distribution in different tumor specimens including whole sections, core needle biopsies, and tissue microarrays. Multiplexed technologies associated with image analysis software can be performed with a high-quality throughput assay to study cancer specimens and are important tools for new discoveries. The different multiplexed technologies described in this review have shown their utility in the study of cancer tissues and their advantages for translational research studies and application in cancer prevention and treatments.
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28
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Gomez-Varela D, Barry AM, Schmidt M. Proteome-based systems biology in chronic pain. J Proteomics 2019; 190:1-11. [DOI: 10.1016/j.jprot.2018.04.004] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2017] [Revised: 03/15/2018] [Accepted: 04/05/2018] [Indexed: 02/07/2023]
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29
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Piga I, Capitoli G, Tettamanti S, Denti V, Smith A, Chinello C, Stella M, Leni D, Garancini M, Galimberti S, Magni F, Pagni F. Feasibility Study for the MALDI-MSI Analysis of Thyroid Fine Needle Aspiration Biopsies: Evaluating the Morphological and Proteomic Stability Over Time. Proteomics Clin Appl 2018; 13:e1700170. [PMID: 30411853 DOI: 10.1002/prca.201700170] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2018] [Revised: 10/18/2018] [Indexed: 11/05/2022]
Abstract
PURPOSE MALDI-MS imaging (MALDI-MSI) is an emerging technology that enables the spatial distribution of biomolecules within tissue to be combined with the traditional morphological information familiar to clinicians. Thus, for diagnostic or prognostic purposes, along with predicting response to therapeutic treatment, it is important to properly collect and handle biological specimens in order to avoid degradation or the formation of artifacts in the morphological structure and proteomic profile. EXPERIMENTAL DESIGN In this work, the morphological and proteomic stability of thyroid fine needle aspiration biopsies in PreservCyt (up to 14 days) and CytoLyt (up to 7 days) solutions at 4 °C has been verified, by MALDI-MSI analysis. Moreover, a new measure has been introduced in order to assess the similarity of the obtained MALDI-MSI spectra, by equally taking into account the number of signals (fit and retrofit), and their intensities (Spearman's correlation and spectra overlap). RESULTS Results show no degradation of the cellular morphology and a good stability of the samples up to 14 days in PreservCyt solution. CONCLUSIONS AND CLINICAL RELEVANCE Moreover, this protocol can be easily implemented in pathological units, allowing simple sample collection and shipment to be used not only for the proteomic MALDI-MSI analysis of thyroid FNABs but also for other biological liquid based specimens.
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Affiliation(s)
- Isabella Piga
- Department of Medicine and Surgery, Clinical Proteomics and Metabolomics Unit, University of Milano-Bicocca, Vedano al Lambro, Italy.,Department of Medicine and Surgery, Section of Pathology, University of Milano-Bicocca, Monza, Italy
| | - Giulia Capitoli
- Department of Medicine and Surgery, Centre of Biostatistics for Clinical Epidemiology, University of Milano-Bicocca, Monza, Italy
| | - Silvia Tettamanti
- Department of Medicine and Surgery, Clinical Proteomics and Metabolomics Unit, University of Milano-Bicocca, Vedano al Lambro, Italy
| | - Vanna Denti
- Department of Medicine and Surgery, Clinical Proteomics and Metabolomics Unit, University of Milano-Bicocca, Vedano al Lambro, Italy
| | - Andrew Smith
- Department of Medicine and Surgery, Clinical Proteomics and Metabolomics Unit, University of Milano-Bicocca, Vedano al Lambro, Italy
| | - Clizia Chinello
- Department of Medicine and Surgery, Clinical Proteomics and Metabolomics Unit, University of Milano-Bicocca, Vedano al Lambro, Italy
| | - Martina Stella
- Department of Medicine and Surgery, Clinical Proteomics and Metabolomics Unit, University of Milano-Bicocca, Vedano al Lambro, Italy
| | - Davide Leni
- Department of Radiology, San Gerardo Hospital, Monza, Italy
| | | | - Stefania Galimberti
- Department of Medicine and Surgery, Centre of Biostatistics for Clinical Epidemiology, University of Milano-Bicocca, Monza, Italy
| | - Fulvio Magni
- Department of Medicine and Surgery, Clinical Proteomics and Metabolomics Unit, University of Milano-Bicocca, Vedano al Lambro, Italy
| | - Fabio Pagni
- Department of Medicine and Surgery, Section of Pathology, University of Milano-Bicocca, Monza, Italy
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30
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Titz B, Gadaleta RM, Lo Sasso G, Elamin A, Ekroos K, Ivanov NV, Peitsch MC, Hoeng J. Proteomics and Lipidomics in Inflammatory Bowel Disease Research: From Mechanistic Insights to Biomarker Identification. Int J Mol Sci 2018; 19:ijms19092775. [PMID: 30223557 PMCID: PMC6163330 DOI: 10.3390/ijms19092775] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2018] [Revised: 09/11/2018] [Accepted: 09/12/2018] [Indexed: 02/06/2023] Open
Abstract
Inflammatory bowel disease (IBD) represents a group of progressive disorders characterized by recurrent chronic inflammation of the gut. Ulcerative colitis and Crohn's disease are the major manifestations of IBD. While our understanding of IBD has progressed in recent years, its etiology is far from being fully understood, resulting in suboptimal treatment options. Complementing other biological endpoints, bioanalytical "omics" methods that quantify many biomolecules simultaneously have great potential in the dissection of the complex pathogenesis of IBD. In this review, we focus on the rapidly evolving proteomics and lipidomics technologies and their broad applicability to IBD studies; these range from investigations of immune-regulatory mechanisms and biomarker discovery to studies dissecting host⁻microbiome interactions and the role of intestinal epithelial cells. Future studies can leverage recent advances, including improved analytical methodologies, additional relevant sample types, and integrative multi-omics analyses. Proteomics and lipidomics could effectively accelerate the development of novel targeted treatments and the discovery of complementary biomarkers, enabling continuous monitoring of the treatment response of individual patients; this may allow further refinement of treatment and, ultimately, facilitate a personalized medicine approach to IBD.
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Affiliation(s)
- Bjoern Titz
- PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchatel, Switzerland.
| | - Raffaella M Gadaleta
- PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchatel, Switzerland.
| | - Giuseppe Lo Sasso
- PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchatel, Switzerland.
| | - Ashraf Elamin
- PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchatel, Switzerland.
| | - Kim Ekroos
- Lipidomics Consulting Ltd., Irisviksvägen 31D, 02230 Esbo, Finland.
| | - Nikolai V Ivanov
- PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchatel, Switzerland.
| | - Manuel C Peitsch
- PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchatel, Switzerland.
| | - Julia Hoeng
- PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, CH-2000 Neuchatel, Switzerland.
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31
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Zagorac I, Fernandez-Gaitero S, Penning R, Post H, Bueno MJ, Mouron S, Manso L, Morente MM, Alonso S, Serra V, Muñoz J, Gómez-López G, Lopez-Acosta JF, Jimenez-Renard V, Gris-Oliver A, Al-Shahrour F, Piñeiro-Yañez E, Montoya-Suarez JL, Apala JV, Moreno-Torres A, Colomer R, Dopazo A, Heck AJR, Altelaar M, Quintela-Fandino M. In vivo phosphoproteomics reveals kinase activity profiles that predict treatment outcome in triple-negative breast cancer. Nat Commun 2018; 9:3501. [PMID: 30158526 PMCID: PMC6115463 DOI: 10.1038/s41467-018-05742-z] [Citation(s) in RCA: 42] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2017] [Accepted: 07/19/2018] [Indexed: 01/25/2023] Open
Abstract
Triple-negative breast cancer (TNBC) lacks prognostic and predictive markers. Here, we use high-throughput phosphoproteomics to build a functional TNBC taxonomy. A cluster of 159 phosphosites is upregulated in relapsed cases of a training set (n = 34 patients), with 11 hyperactive kinases accounting for this phosphoprofile. A mass-spectrometry-to-immunohistochemistry translation step, assessing 2 independent validation sets, reveals 6 kinases with preserved independent prognostic value. The kinases split the validation set into two patterns: one without hyperactive kinases being associated with a >90% relapse-free rate, and the other one showing ≥1 hyperactive kinase and being associated with an up to 9.5-fold higher relapse risk. Each kinase pattern encompasses different mutational patterns, simplifying mutation-based taxonomy. Drug regimens designed based on these 6 kinases show promising antitumour activity in TNBC cell lines and patient-derived xenografts. In summary, the present study elucidates phosphosites and kinases implicated in TNBC and suggests a target-based clinical classification system for TNBC. Triple-negative breast cancer (TNBC) lacks prognostic and predictive markers. Here, the authors use phosphoproteomics to define kinases with distinct activity profiles in TNBC, demonstrating their prognostic value as well as their utility for simplifying TNBC classification and designing drug regimens.
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Affiliation(s)
- Ivana Zagorac
- Breast Cancer Clinical Research Unit, CNIO - Spanish National Cancer Research Center, 28029, Madrid, Spain
| | - Sara Fernandez-Gaitero
- Breast Cancer Clinical Research Unit, CNIO - Spanish National Cancer Research Center, 28029, Madrid, Spain
| | - Renske Penning
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.,Netherlands Proteomics Center, Padualaan 8, 3584 CH, Utrecht, The Netherlands
| | - Harm Post
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.,Netherlands Proteomics Center, Padualaan 8, 3584 CH, Utrecht, The Netherlands
| | - Maria J Bueno
- Breast Cancer Clinical Research Unit, CNIO - Spanish National Cancer Research Center, 28029, Madrid, Spain
| | - Silvana Mouron
- Breast Cancer Clinical Research Unit, CNIO - Spanish National Cancer Research Center, 28029, Madrid, Spain
| | - Luis Manso
- Medical Oncology, Hospital 12 de Octubre, Madrid, 28029, Spain
| | - Manuel M Morente
- Biobank, CNIO - Spanish National Cancer Research Center, Madrid, 28029, Spain
| | - Soledad Alonso
- Pathology Department, Hospital Universitario de Guadalajara, Guadalajara, 19002, Spain
| | - Violeta Serra
- Experimental Therapeutics Group, VHIO - Vall d'Hebron Institute of Oncology, Barcelona, 08035, Spain
| | - Javier Muñoz
- Proteomics Unit, CNIO - Spanish National Cancer Research Center, Madrid, 28029, Spain
| | - Gonzalo Gómez-López
- Bioinformatics Unit, CNIO - Spanish National Cancer Research Center, Madrid, 28029, Spain
| | | | - Veronica Jimenez-Renard
- Breast Cancer Clinical Research Unit, CNIO - Spanish National Cancer Research Center, 28029, Madrid, Spain
| | - Albert Gris-Oliver
- Experimental Therapeutics Group, VHIO - Vall d'Hebron Institute of Oncology, Barcelona, 08035, Spain
| | - Fatima Al-Shahrour
- Bioinformatics Unit, CNIO - Spanish National Cancer Research Center, Madrid, 28029, Spain
| | - Elena Piñeiro-Yañez
- Bioinformatics Unit, CNIO - Spanish National Cancer Research Center, Madrid, 28029, Spain
| | | | - Juan V Apala
- Breast Cancer Clinical Research Unit, CNIO - Spanish National Cancer Research Center, 28029, Madrid, Spain
| | - Amalia Moreno-Torres
- Pathology Department, Hospital Universitario de Fuenlabrada, Fuenlabrada, 28942, Spain
| | - Ramon Colomer
- Medical Oncology, Hospital La Princesa, Madrid, 28006, Spain
| | - Ana Dopazo
- Genomics Unit, CNIC - Spanish National Center for Cardiovascular Research, Madrid, 28029, Spain
| | - Albert J R Heck
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.,Netherlands Proteomics Center, Padualaan 8, 3584 CH, Utrecht, The Netherlands
| | - Maarten Altelaar
- Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.,Netherlands Proteomics Center, Padualaan 8, 3584 CH, Utrecht, The Netherlands
| | - Miguel Quintela-Fandino
- Breast Cancer Clinical Research Unit, CNIO - Spanish National Cancer Research Center, 28029, Madrid, Spain. .,Medical Oncology, Hospital Universitario Fuenlabrada, Madrid, 28942, Spain. .,Medical Oncology, Hospital Universitario Quirón, Madrid, 28223, Spain.
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32
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Gaffney EF, Riegman PH, Grizzle WE, Watson PH. Factors that drive the increasing use of FFPE tissue in basic and translational cancer research. Biotech Histochem 2018; 93:373-386. [PMID: 30113239 DOI: 10.1080/10520295.2018.1446101] [Citation(s) in RCA: 80] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
The decision to use 10% neutral buffered formalin fixed, paraffin embedded (FFPE) archival pathology material may be dictated by the cancer research question or analytical technique, or may be governed by national ethical, legal and social implications (ELSI), biobank, and sample availability and access policy. Biobanked samples of common tumors are likely to be available, but not all samples will be annotated with treatment and outcomes data and this may limit their application. Tumors that are rare or very small exist mostly in FFPE pathology archives. Pathology departments worldwide contain millions of FFPE archival samples, but there are challenges to availability. Pathology departments lack resources for retrieving materials for research or for having pathologists select precise areas in paraffin blocks, a critical quality control step. When samples must be sourced from several pathology departments, different fixation and tissue processing approaches create variability in quality. Researchers must decide what sample quality and quality tolerance fit their specific purpose and whether sample enrichment is required. Recent publications report variable success with techniques modified to examine all common species of molecular targets in FFPE samples. Rigorous quality management may be particularly important in sample preparation for next generation sequencing and for optimizing the quality of extracted proteins for proteomics studies. Unpredictable failures, including unpublished ones, likely are related to pre-analytical factors, unstable molecular targets, biological and clinical sampling factors associated with specific tissue types or suboptimal quality management of pathology archives. Reproducible results depend on adherence to pre-analytical phase standards for molecular in vitro diagnostic analyses for DNA, RNA and in particular, extracted proteins. With continuing adaptations of techniques for application to FFPE, the potential to acquire much larger numbers of FFPE samples and the greater convenience of using FFPE in assays for precision medicine, the choice of material in the future will become increasingly biased toward FFPE samples from pathology archives. Recognition that FFPE samples may harbor greater variation in quality than frozen samples for several reasons, including variations in fixation and tissue processing, requires that FFPE results be validated provided a cohort of frozen tissue samples is available.
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Affiliation(s)
- E F Gaffney
- a Biobank Ireland Trust , Malahide , Co Dublin , Ireland
| | - P H Riegman
- b Erasmus Medical Centre , Department of Pathology , Rotterdam , The Netherlands
| | - W E Grizzle
- c Department of Pathology , University of Alabama at Birmingham (UAB) , Birmingham , Alabama , USA
| | - P H Watson
- d BC Cancer Agency , Vancouver Island Center , Victoria , BC , Canada
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Piehowski PD, Petyuk VA, Sontag RL, Gritsenko MA, Weitz KK, Fillmore TL, Moon J, Makhlouf H, Chuaqui RF, Boja ES, Rodriguez H, Lee JSH, Smith RD, Carrick DM, Liu T, Rodland KD. Residual tissue repositories as a resource for population-based cancer proteomic studies. Clin Proteomics 2018; 15:26. [PMID: 30087585 PMCID: PMC6074037 DOI: 10.1186/s12014-018-9202-4] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2018] [Accepted: 07/27/2018] [Indexed: 11/26/2022] Open
Abstract
BACKGROUND Mass spectrometry-based proteomics has become a powerful tool for the identification and quantification of proteins from a wide variety of biological specimens. To date, the majority of studies utilizing tissue samples have been carried out on prospectively collected fresh frozen or optimal cutting temperature (OCT) embedded specimens. However, such specimens are often difficult to obtain, in limited in supply, and clinical information and outcomes on patients are inherently delayed as compared to banked samples. Annotated formalin fixed, paraffin embedded (FFPE) tumor tissue specimens are available for research use from a variety of tissue banks, such as from the surveillance, epidemiology and end results (SEER) registries' residual tissue repositories. Given the wealth of outcomes information associated with such samples, the reuse of archived FFPE blocks for deep proteomic characterization with mass spectrometry technologies would provide a valuable resource for population-based cancer studies. Further, due to the widespread availability of FFPE specimens, validation of specimen integrity opens the possibility for thousands of studies that can be conducted worldwide. METHODS To examine the suitability of the SEER repository tissues for proteomic and phosphoproteomic analysis, we analyzed 60 SEER patient samples, with time in storage ranging from 7 to 32 years; 60 samples with expression proteomics and 18 with phosphoproteomics, using isobaric labeling. Linear modeling and gene set enrichment analysis was used to evaluate the impacts of collection site and storage time. RESULTS All samples, regardless of age, yielded suitable protein mass after extraction for expression analysis and 18 samples yielded sufficient mass for phosphopeptide analysis. Although peptide, protein, and phosphopeptide identifications were reduced by 50, 20 and 76% respectively, from comparable OCT specimens, we found no statistically significant differences in protein quantitation correlating with collection site or specimen age. GSEA analysis of GO-term level measurements of protein abundance differences between FFPE and OCT embedded specimens suggest that the formalin fixation process may alter representation of protein categories in the resulting dataset. CONCLUSIONS These studies demonstrate that residual FFPE tissue specimens, of varying age and collection site, are a promising source of protein for proteomic investigations if paired with rigorously verified mass spectrometry workflows.
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Affiliation(s)
- Paul D. Piehowski
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354 USA
| | - Vladislav A. Petyuk
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354 USA
| | - Ryan L. Sontag
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354 USA
| | - Marina A. Gritsenko
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354 USA
| | - Karl K. Weitz
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354 USA
| | - Thomas L. Fillmore
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354 USA
| | - Jamie Moon
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354 USA
| | - Hala Makhlouf
- Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, MD 20850 USA
| | - Rodrigo F. Chuaqui
- Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, MD 20850 USA
| | - Emily S. Boja
- Office of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892 USA
| | - Henry Rodriguez
- Office of Cancer Clinical Proteomics Research, National Cancer Institute, Bethesda, MD 20892 USA
| | - Jerry S. H. Lee
- Center for Strategic Scientific Initiatives, National Cancer Institute, Bethesda, MD 20892 USA
| | - Richard D. Smith
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354 USA
| | - Danielle M. Carrick
- Division of Cancer Control and Population Sciences, National Cancer Institute, Rockville, MD 20850 USA
| | - Tao Liu
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354 USA
| | - Karin D. Rodland
- Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354 USA
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Tutturen AEV, Dørum S, Clancy T, Reims HM, Christophersen A, Lundin KEA, Sollid LM, de Souza GA, Stamnaes J. Characterization of the Small Intestinal Lesion in Celiac Disease by Label-Free Quantitative Mass Spectrometry. THE AMERICAN JOURNAL OF PATHOLOGY 2018; 188:1563-1579. [PMID: 29684362 DOI: 10.1016/j.ajpath.2018.03.017] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/21/2017] [Revised: 02/08/2018] [Accepted: 03/13/2018] [Indexed: 12/16/2022]
Abstract
Global characterization of tissue proteomes from small amounts of biopsy material has become feasible because of advances in mass spectrometry and bioinformatics tools. In celiac disease (CD), dietary gluten induces an immune response that is accompanied by pronounced remodeling of the small intestine. Removal of gluten from the diet abrogates the immune response, and the tissue architecture normalizes. In this study, differences in global protein expression of small intestinal biopsy specimens from CD patients were quantified by analyzing formalin-fixed, paraffin-embedded material using liquid chromatography-mass spectrometry and label-free protein quantitation. Protein expression was compared in biopsy specimens collected from the same patients before and after 1-year treatment with gluten-free diet (n = 10) or before and after 3-day gluten provocation (n = 4). Differential expression of proteins in particular from mature enterocytes, neutrophils, and plasma cells could distinguish untreated from treated CD mucosa, and Ig variable region IGHV5-51 expression was found to serve as a CD-specific marker of ongoing immune activation. In patients who had undergone gluten challenge, coordinated up-regulation of wound response proteins, including the CD autoantigen transglutaminase 2, was observed. Our study provides a global and unbiased assessment of antigen-driven changes in protein expression in the celiac intestinal mucosa.
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Affiliation(s)
- Astrid E V Tutturen
- Centre for Immune Regulation and Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway; Proteomics Core Facility, Oslo University Hospital-Rikshospitalet, Oslo, Norway
| | - Siri Dørum
- Centre for Immune Regulation and Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway
| | - Trevor Clancy
- Department of Immunology, Institute for Cancer Research, Oslo University Hospital-Radiumhospitalet, Oslo, Norway
| | - Henrik M Reims
- Department of Pathology, Oslo University Hospital, Oslo, Norway
| | | | - Knut E A Lundin
- KG Jebsen Coeliac Disease Research Centre, University of Oslo, Oslo, Norway; Department of Gastroenterology, Oslo University Hospital-Rikshospitalet, Oslo, Norway
| | - Ludvig M Sollid
- Centre for Immune Regulation and Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway; KG Jebsen Coeliac Disease Research Centre, University of Oslo, Oslo, Norway
| | - Gustavo A de Souza
- Proteomics Core Facility, Oslo University Hospital-Rikshospitalet, Oslo, Norway; The Brain Institute, Universidade Federal do Rio Grande do Norte, Natal-RN, Brazil
| | - Jorunn Stamnaes
- Centre for Immune Regulation and Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway.
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35
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Ongay S, Langelaar-Makkinje M, Stoop MP, Liu N, Overkleeft H, Luider TM, Groothuis GMM, Bischoff R. Cleavable Crosslinkers as Tissue Fixation Reagents for Proteomic Analysis. Chembiochem 2018; 19:736-743. [PMID: 29356267 DOI: 10.1002/cbic.201700625] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2017] [Indexed: 12/17/2022]
Abstract
Formaldehyde fixation is widely used for long-term maintenance of tissue. However, due to formaldehyde-induced crosslinks, fixed tissue proteins are difficult to extract, which hampers mass spectrometry (MS) proteomic analyses. Recent years have seen the use of different combinations of high temperature and solubilizing agents (usually derived from antigen retrieval techniques) to unravel formaldehyde-fixed paraffin-embedded tissue proteomes. However, to achieve protein extraction yields similar to those of fresh-frozen tissue, high-temperature heating is necessary. Such harsh extraction conditions can affect sensitive amino acids and post-translational modifications, resulting in the loss of important information, while still not resulting in protein yields comparable to those of fresh-frozen tissue. Herein, the objective is to evaluate cleavable protein crosslinkers as fixatives that allow tissue preservation and efficient protein extraction from fixed tissue for MS proteomics under mild conditions. With this goal in mind, disuccinimidyl tartrate (DST) and dithiobis(succinimidylpropionate) (DSP) are investigated as cleavable fixating reagents. These compounds crosslink proteins by reacting with amino groups, leading to amide bond formation, and can be cleaved with sodium metaperiodate (cis-diols, DST) or reducing agents (disulfide bonds, DSP), respectively. Results show that cleavable protein crosslinking with DST and DSP allows tissue fixation with morphology preservation comparable to that of formaldehyde. In addition, cleavage of DSP improves protein recovery from fixed tissue by a factor of 18 and increases the number of identified proteins by approximately 20 % under mild extraction conditions compared with those of formaldehyde-fixed paraffin-embedded tissue. A major advantage of DSP is the introduction of well-defined protein modifications that can be taken into account during database searching. In contrast to DSP fixation, DST fixation followed by cleavage with sodium metaperiodate, although effective, results in side reactions that prevent effective protein extraction and interfere with protein identification. Protein crosslinkers that can be cleaved under mild conditions and result in defined modifications, such as DSP, are thus viable alternatives to formaldehyde as tissue fixatives to facilitate protein analysis from paraffin-embedded, fixed tissue.
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Affiliation(s)
- Sara Ongay
- Department of Analytical Biochemistry, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands
| | - Miriam Langelaar-Makkinje
- Department Pharmacokinetics, Toxicology and Targeting, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands
| | - Marcel P Stoop
- Department of Neurology, Erasmus University Medical Center, P. O. Box 1738, 3000 DR, Rotterdam, The Netherlands
| | - Nora Liu
- Department of Bio-Organic Synthesis, Leiden University, P. O. Box 9502, 2300 RA, Leiden, The Netherlands
| | - Hermen Overkleeft
- Department of Bio-Organic Synthesis, Leiden University, P. O. Box 9502, 2300 RA, Leiden, The Netherlands
| | - Theo M Luider
- Department of Neurology, Erasmus University Medical Center, P. O. Box 1738, 3000 DR, Rotterdam, The Netherlands
| | - Geny M M Groothuis
- Department Pharmacokinetics, Toxicology and Targeting, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands
| | - Rainer Bischoff
- Department of Analytical Biochemistry, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, The Netherlands
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Zhao T, Wu J, Liu X, Zhang L, Chen G, Lu H. Diagnosis of thymic epithelial tumor subtypes by a quantitative proteomic approach. Analyst 2018; 143:2491-2500. [PMID: 29645059 DOI: 10.1039/c8an00218e] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
This study shows the first depth proteomic profiling of all TET subtypes and six candidate biomarkers were identified and validated.
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Affiliation(s)
- Ting Zhao
- Institutes of Biomedical Sciences and Department of Chemistry
- Fudan University
- Shanghai 200032
- P.R. China
| | - Jie Wu
- Department of Pathology
- Zhongshan Hospital
- Fudan University
- Shanghai 200032
- P.R. China
| | - Xiaohui Liu
- Institutes of Biomedical Sciences and Department of Chemistry
- Fudan University
- Shanghai 200032
- P.R. China
| | - Lei Zhang
- Institutes of Biomedical Sciences and Department of Chemistry
- Fudan University
- Shanghai 200032
- P.R. China
| | - Gang Chen
- Department of Pathology
- Zhongshan Hospital
- Fudan University
- Shanghai 200032
- P.R. China
| | - Haojie Lu
- Institutes of Biomedical Sciences and Department of Chemistry
- Fudan University
- Shanghai 200032
- P.R. China
- Key Laboratory of Glycoconjugates Research Ministry of Public Health
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37
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Herfs M, Longuespée R, Quick CM, Roncarati P, Suarez-Carmona M, Hubert P, Lebeau A, Bruyere D, Mazzucchelli G, Smargiasso N, Baiwir D, Lai K, Dunn A, Obregon F, Yang EJ, Pauw ED, Crum CP, Delvenne P. Proteomic signatures reveal a dualistic and clinically relevant classification of anal canal carcinoma. J Pathol 2017; 241:522-533. [DOI: 10.1002/path.4858] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2016] [Revised: 11/21/2016] [Accepted: 11/30/2016] [Indexed: 12/11/2022]
Affiliation(s)
- Michael Herfs
- Laboratory of Experimental Pathology, GIGA-Cancer; University of Liège; Liège Belgium
| | - Rémi Longuespée
- Mass Spectrometry Laboratory, Systems Biology and Chemical Biology, GIGA-Research; University of Liège; Liège Belgium
| | - Charles M Quick
- Department of Pathology; University of Arkansas for Medical Sciences; Little Rock AR USA
| | - Patrick Roncarati
- Laboratory of Experimental Pathology, GIGA-Cancer; University of Liège; Liège Belgium
| | - Meggy Suarez-Carmona
- Laboratory of Experimental Pathology, GIGA-Cancer; University of Liège; Liège Belgium
| | - Pascale Hubert
- Laboratory of Experimental Pathology, GIGA-Cancer; University of Liège; Liège Belgium
| | - Alizée Lebeau
- Laboratory of Experimental Pathology, GIGA-Cancer; University of Liège; Liège Belgium
| | - Diane Bruyere
- Laboratory of Experimental Pathology, GIGA-Cancer; University of Liège; Liège Belgium
| | - Gabriel Mazzucchelli
- Mass Spectrometry Laboratory, Systems Biology and Chemical Biology, GIGA-Research; University of Liège; Liège Belgium
| | - Nicolas Smargiasso
- Mass Spectrometry Laboratory, Systems Biology and Chemical Biology, GIGA-Research; University of Liège; Liège Belgium
| | - Dominique Baiwir
- Mass Spectrometry Laboratory, Systems Biology and Chemical Biology, GIGA-Research; University of Liège; Liège Belgium
- GIGA Proteomic Facility; University of Liège; Liège Belgium
| | - Keith Lai
- Department of Pathology; University of Arkansas for Medical Sciences; Little Rock AR USA
| | - Andrew Dunn
- Department of Pathology; University of Arkansas for Medical Sciences; Little Rock AR USA
| | - Fabiola Obregon
- Department of Pathology; University of Arkansas for Medical Sciences; Little Rock AR USA
| | - Eric J Yang
- Department of Pathology, Division of Women's and Perinatal Pathology, Brigham and Women's Hospital; Harvard Medical School; Boston MA USA
| | - Edwin De Pauw
- Mass Spectrometry Laboratory, Systems Biology and Chemical Biology, GIGA-Research; University of Liège; Liège Belgium
| | - Christopher P Crum
- Department of Pathology, Division of Women's and Perinatal Pathology, Brigham and Women's Hospital; Harvard Medical School; Boston MA USA
| | - Philippe Delvenne
- Laboratory of Experimental Pathology, GIGA-Cancer; University of Liège; Liège Belgium
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38
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Psatha K, Kollipara L, Voutyraki C, Divanach P, Sickmann A, Rassidakis GZ, Drakos E, Aivaliotis M. Deciphering lymphoma pathogenesis via state-of-the-art mass spectrometry-based quantitative proteomics. J Chromatogr B Analyt Technol Biomed Life Sci 2016; 1047:2-14. [PMID: 27979587 DOI: 10.1016/j.jchromb.2016.11.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2016] [Revised: 10/18/2016] [Accepted: 11/04/2016] [Indexed: 12/13/2022]
Abstract
Mass spectrometry-based quantitative proteomics specifically applied to comprehend the pathogenesis of lymphoma has incremental value in deciphering the heterogeneity in complex deregulated molecular mechanisms/pathways of the lymphoma entities, implementing the current diagnostic and therapeutic strategies. Essential global, targeted and functional differential proteomics analyses although still evolving, have been successfully implemented to shed light on lymphoma pathogenesis to discover and explore the role of potential lymphoma biomarkers and drug targets. This review aims to outline and appraise the present status of MS-based quantitative proteomic approaches in lymphoma research, introducing the current state-of-the-art MS-based proteomic technologies, the opportunities they offer in biological discovery in human lymphomas and the related limitation issues arising from sample preparation to data evaluation. It is a synopsis containing information obtained from recent research articles, reviews and public proteomics repositories (PRIDE). We hope that this review article will aid, assimilate and assess all the information aiming to accelerate the development and validation of diagnostic, prognostic or therapeutic targets for an improved and empowered clinical proteomics application in lymphomas in the nearby future.
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Affiliation(s)
- Konstantina Psatha
- Institute of Molecular Biology and Biotechnology, FORTH, Heraklion, Greece; School of Medicine, National and Kapodistrian University of Athens, Athens, Greece; Department of Pathology, School of Medicine, University of Crete, Heraklion, Greece
| | - Laxmikanth Kollipara
- Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany
| | | | - Peter Divanach
- Institute of Molecular Biology and Biotechnology, FORTH, Heraklion, Greece
| | - Albert Sickmann
- Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany; Department of Chemistry, College of Physical Sciences, University of Aberdeen, Aberdeen, Scotland, United Kingdom; Medizinische Fakultät, Medizinische Proteom-Center (MPC), Ruhr-Universität Bochum, Bochum, Germany
| | - George Z Rassidakis
- School of Medicine, National and Kapodistrian University of Athens, Athens, Greece; Department of Pathology and Cytology, Karolinska University Hospital and Karolinska Institute, Radiumhemmet, Stockholm, SE-17176, Sweden
| | - Elias Drakos
- Department of Pathology, School of Medicine, University of Crete, Heraklion, Greece
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39
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Kennedy JJ, Whiteaker JR, Schoenherr RM, Yan P, Allison K, Shipley M, Lerch M, Hoofnagle AN, Baird GS, Paulovich AG. Optimized Protocol for Quantitative Multiple Reaction Monitoring-Based Proteomic Analysis of Formalin-Fixed, Paraffin-Embedded Tissues. J Proteome Res 2016; 15:2717-28. [PMID: 27462933 DOI: 10.1021/acs.jproteome.6b00245] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R(2) = 0.94) and immuno-MRM (R(2) = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens.
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Affiliation(s)
- Jacob J Kennedy
- Clinical Research Division, Fred Hutchinson Cancer Research Center , Seattle, Washington 98109, United States
| | - Jeffrey R Whiteaker
- Clinical Research Division, Fred Hutchinson Cancer Research Center , Seattle, Washington 98109, United States
| | - Regine M Schoenherr
- Clinical Research Division, Fred Hutchinson Cancer Research Center , Seattle, Washington 98109, United States
| | - Ping Yan
- Clinical Research Division, Fred Hutchinson Cancer Research Center , Seattle, Washington 98109, United States
| | - Kimberly Allison
- Department of Pathology, Stanford University , Stanford, California 94305 United States
| | - Melissa Shipley
- Department of Laboratory Medicine, University of Washington , Seattle, Washington 98195 United States
| | - Melissa Lerch
- Department of Laboratory Medicine, University of Washington , Seattle, Washington 98195 United States
| | - Andrew N Hoofnagle
- Department of Laboratory Medicine, University of Washington , Seattle, Washington 98195 United States
| | - Geoffrey Stuart Baird
- Department of Laboratory Medicine, University of Washington , Seattle, Washington 98195 United States
| | - Amanda G Paulovich
- Clinical Research Division, Fred Hutchinson Cancer Research Center , Seattle, Washington 98109, United States
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40
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Mathieson W, Marcon N, Antunes L, Ashford DA, Betsou F, Frasquilho SG, Kofanova OA, McKay SC, Pericleous S, Smith C, Unger KM, Zeller C, Thomas GA. A Critical Evaluation of the PAXgene Tissue Fixation System: Morphology, Immunohistochemistry, Molecular Biology, and Proteomics. Am J Clin Pathol 2016; 146:25-40. [PMID: 27402607 DOI: 10.1093/ajcp/aqw023] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
OBJECTIVES To evaluate the PAXgene tissue fixation system. METHODS Clinical biospecimens (n = 46) were divided into PAXgene-fixed paraffin-embedded (PFPE), formalin-fixed paraffin-embedded (FFPE), and fresh-frozen (FF) blocks. PFPE and FFPE sections were compared for histology (H&E staining) and immunohistochemistry (14 antibodies) using tissue microarrays. PFPE, FFPE, and FF samples were compared in terms of RNA quality (RNA integrity number, polymerase chain reaction [PCR] amplicon length, and quantitative reverse transcription PCR), DNA quality (gel electrophoresis and methylation profiling) and protein quality (liquid chromatography-mass spectrometry [LC-MS/MS]). RESULTS PFPE protocol optimization was required in most cases and is described. RNA extracted from PFPE sections was considerably less degraded than that from FFPE sections but more degraded than that from FF blocks. Genomic-length DNA was extracted from PFPE and FF biospecimens, and methylation profiling showed PFPE and FF biospecimens to be almost indistinguishable. Only degraded DNA was extracted from FFPE biospecimens. PFPE sections yielded peptides that were slightly less amenable to LC-MS/MS analysis than FFPE sections, but FF gave slightly better results. CONCLUSIONS While it cannot be envisaged that PAXgene will replace formalin in a routine clinical setting, for specific projects or immunodiagnostics involving biospecimens destined for immunohistochemical or histologic staining and DNA or RNA analyses, PAXgene is a viable option.
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Affiliation(s)
- William Mathieson
- From the Integrated Biobank of Luxembourg, Luxembourg
- Department of Surgery and Cancer, Imperial College London, London, United Kingdom
| | | | | | - David A. Ashford
- Bioscience Technology Facility, Department of Biology, University of York, York, United Kingdom
| | - Fay Betsou
- From the Integrated Biobank of Luxembourg, Luxembourg
| | | | | | - Siobhan C. McKay
- Department of Surgery and Cancer, Imperial College London, London, United Kingdom
| | - Stephan Pericleous
- Department of Surgery and Cancer, Imperial College London, London, United Kingdom
| | - Colleen Smith
- Wales Cancer Bank, Singleton Hospital, Swansea, United Kingdom
| | - Kristian M. Unger
- Department of Surgery and Cancer, Imperial College London, London, United Kingdom
| | - Constanze Zeller
- Department of Surgery and Cancer, Imperial College London, London, United Kingdom
| | - Geraldine A. Thomas
- Department of Surgery and Cancer, Imperial College London, London, United Kingdom
- Wales Cancer Bank, Singleton Hospital, Swansea, United Kingdom
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Bennike TB, Kastaniegaard K, Padurariu S, Gaihede M, Birkelund S, Andersen V, Stensballe A. ⿿Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human tissue samples. EUPA OPEN PROTEOMICS 2016; 10:9-18. [PMID: 29900094 PMCID: PMC5988570 DOI: 10.1016/j.euprot.2015.10.001] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/24/2015] [Revised: 08/19/2015] [Accepted: 10/25/2015] [Indexed: 01/14/2023]
Abstract
Large biobanks exist worldwide containing formalin-fixed, paraffin-embedded samples and samples stored in RNAlater. However, the impact of tissue preservation on the result of a quantative proteome analysis remains poorly described. Human colon mucosal biopsies were extracted from the sigmoideum and either immediately frozen, stabilized in RNAlater, or stabilized by formalin-fixation. In one set of biopsies, formalin stabilization was delayed for 30 min. The protein content of the samples was characterized by high throughput quantitative proteomics. We were able to identify a similar high number of proteins in the samples regardless of preservation method, with only minor differences in protein quantitation.
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Key Words
- CAN, acetonitrile
- DF, directly-frozen
- FA, formic acid
- FASP, filter-aided sample preparation
- FDR, false discovery rate
- FFPE, formalin-fixed
- Formalin-fixed
- HLA-A class I, histocompatibility antigen A-23 alpha chain
- HLA-DRB1 class II, histocompatibility antigen DRB1-4 beta chain
- Human colon mucosa
- LFQ, label-free quantification
- Mass spectrometry
- PCA, principle component analysis
- PSM, peptide spectral match
- PTM, post-translational modification
- Paraffin-embedded
- Preservation
- Proteomics
- RNAlater
- SDC, sodium deoxycholate
- SDS, sodium dodecyl sulfate
- TEAB, triethylammonium bicarbonate
- iFFPE, immediately formalin-fixed
- s, standard deviation
- sFFPE, stored for 30 min prior to formalin-fixed
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Affiliation(s)
- Tue Bjerg Bennike
- Research Unit for Molecular Diagnostic and Clinical Research, Hospital of Southern Jutland, Aabenraa, Denmark
- Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| | | | - Simona Padurariu
- Department of Otolaryngology, Head and Neck Surgery, Aalborg University Hospital, Aalborg, Denmark
| | - Michael Gaihede
- Department of Otolaryngology, Head and Neck Surgery, Aalborg University Hospital, Aalborg, Denmark
- Department of Clinical Medicine, Aalborg University, Aalborg, Denmark
| | - Svend Birkelund
- Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
| | - Vibeke Andersen
- Research Unit for Molecular Diagnostic and Clinical Research, Hospital of Southern Jutland, Aabenraa, Denmark
- Institute of Regional Health Research-Center Soenderjylland, University of Southern Denmark, Odense, Denmark
| | - Allan Stensballe
- Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
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Wojakowska A, Marczak Ł, Jelonek K, Polanski K, Widlak P, Pietrowska M. An Optimized Method of Metabolite Extraction from Formalin-Fixed Paraffin-Embedded Tissue for GC/MS Analysis. PLoS One 2015; 10:e0136902. [PMID: 26348873 PMCID: PMC4562636 DOI: 10.1371/journal.pone.0136902] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2015] [Accepted: 08/09/2015] [Indexed: 12/29/2022] Open
Abstract
Formalin-fixed paraffin-embedded (FFPE) tissue specimens constitute a highly valuable source of clinical material for retrospective molecular studies. However, metabolomic assessment of such archival material remains still in its infancy. Hence, there is an urgent need for efficient methods enabling extraction and profiling of metabolites present in FFPE tissue specimens. Here we demonstrate the methodology for isolation of primary metabolites from archival tissues; either fresh-frozen, formalin-fixed or formalin-fixed and paraffin-embedded specimens of mouse kidney were analysed and compared in this work. We used gas chromatography followed by mass spectrometry (GC/MS approach) to identify about 80 metabolites (including amino acids, saccharides, carboxylic acids, fatty acids) present in such archive material. Importantly, about 75% of identified compounds were detected in all three types of specimens. Moreover, we observed that fixation with formalin itself (and their duration) did not affect markedly the presence of particular metabolites in tissue-extracted material, yet fixation for 24h could be recommended as a practical standard. Paraffin embedding influenced efficiency of extraction, which resulted in reduced quantities of several compounds. Nevertheless, we proved applicability of FFPE specimens for non-targeted GS/MS-based profiling of tissue metabolome, which is of great importance for feasibility of metabolomics studies using retrospective clinical material.
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Affiliation(s)
- Anna Wojakowska
- Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska—Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 15, 44–100, Gliwice, Poland
| | - Łukasz Marczak
- Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12/14, 61–704 Poznan, Poland
| | - Karol Jelonek
- Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska—Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 15, 44–100, Gliwice, Poland
| | - Krzysztof Polanski
- Warwick Systems Biology Centre, University of Warwick, Coventry, CV4 7AL, United Kingdom
| | - Piotr Widlak
- Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska—Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 15, 44–100, Gliwice, Poland
| | - Monika Pietrowska
- Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska—Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 15, 44–100, Gliwice, Poland
- * E-mail:
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Steiner C, Tille JC, Lamerz J, Kux van Geijtenbeek S, McKee TA, Venturi M, Rubbia-Brandt L, Hochstrasser D, Cutler P, Lescuyer P, Ducret A. Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues. Mol Cell Proteomics 2015; 14:2786-99. [PMID: 26149442 DOI: 10.1074/mcp.o115.049049] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2015] [Indexed: 11/06/2022] Open
Abstract
The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.
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Affiliation(s)
- Carine Steiner
- From the ‡Division of Laboratory Medicine, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland; §Translational Technologies and Bioinformatics, Pharmaceutical Sciences, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4070 Basel, Switzerland;
| | - Jean-Christophe Tille
- ¶Division of Clinical Pathology, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland
| | - Jens Lamerz
- §Translational Technologies and Bioinformatics, Pharmaceutical Sciences, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4070 Basel, Switzerland
| | - Sabine Kux van Geijtenbeek
- §Translational Technologies and Bioinformatics, Pharmaceutical Sciences, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4070 Basel, Switzerland
| | - Thomas A McKee
- ¶Division of Clinical Pathology, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland
| | - Miro Venturi
- ‖Oncology Division, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Penzberg, Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany
| | - Laura Rubbia-Brandt
- ¶Division of Clinical Pathology, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland
| | - Denis Hochstrasser
- From the ‡Division of Laboratory Medicine, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland
| | - Paul Cutler
- §Translational Technologies and Bioinformatics, Pharmaceutical Sciences, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4070 Basel, Switzerland
| | - Pierre Lescuyer
- From the ‡Division of Laboratory Medicine, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, CH-1211 Geneva, Switzerland
| | - Axel Ducret
- §Translational Technologies and Bioinformatics, Pharmaceutical Sciences, Roche Pharma Research & Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4070 Basel, Switzerland
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Srinivasa S, Ding X, Kast J. Formaldehyde cross-linking and structural proteomics: Bridging the gap. Methods 2015; 89:91-8. [PMID: 25979347 DOI: 10.1016/j.ymeth.2015.05.006] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2015] [Revised: 04/30/2015] [Accepted: 05/06/2015] [Indexed: 12/21/2022] Open
Abstract
Proteins are dynamic entities constantly moving and altering their structures based on their functions and interactions inside and outside the cell. Formaldehyde cross-linking combined with mass spectrometry can accurately capture interactions of these rapidly changing biomolecules while maintaining their physiological surroundings. Even with its numerous established uses in biology and compatibility with mass spectrometry, formaldehyde has not yet been applied in structural proteomics. However, formaldehyde cross-linking is moving toward analyzing tertiary structure, which conventional cross-linkers have already accomplished. The purpose of this review is to describe the potential of formaldehyde cross-linking in structural proteomics by highlighting its applications, characteristics and current status in the field.
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Affiliation(s)
- Savita Srinivasa
- The Biomedical Research Centre, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
| | - Xuan Ding
- The Biomedical Research Centre, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Institute for Genomics, Biocomputing and Biotechnology, Mississippi State University, Mississippi State, MS 39762, United States
| | - Juergen Kast
- The Biomedical Research Centre, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z3, Canada; Centre for Blood Research, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
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Silvestre DD, Brambilla F, Motta S, Mauri P. Evaluation of Proteomic Data: From Profiling to Network Analysis by Way of Biomarker Discovery. BIOMARKER VALIDATION 2015:163-182. [DOI: 10.1002/9783527680658.ch9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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Qualitative and quantitative proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue. Methods Mol Biol 2015; 1295:109-15. [PMID: 25820718 DOI: 10.1007/978-1-4939-2550-6_10] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Formalin-fixed, paraffin-embedded (FFPE) tissue has recently gained interest as an alternative to fresh/frozen tissue for retrospective protein biomarker discovery. However, during the formalin fixation proteins undergo degradation and cross-linking, making conventional protein analysis technologies challenging. Cross-linking is even more challenging when quantitative proteome analysis of FFPE tissue is planned. The use of conventional protein labeling technologies on FFPE tissue has turned out to be problematic as the lysine residue labeling targets are frequently blocked by the formalin treatment. We have established a qualitative and quantitative proteomics analysis technique for FFPE tissues that combines label-free proteomic analysis with optimized protein extraction and separation conditions.
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Aichler M, Luber B, Lordick F, Walch A. Proteomic and metabolic prediction of response to therapy in gastric cancer. World J Gastroenterol 2014; 20:13648-13657. [PMID: 25320503 PMCID: PMC4194549 DOI: 10.3748/wjg.v20.i38.13648] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/09/2013] [Accepted: 06/13/2014] [Indexed: 02/06/2023] Open
Abstract
Several new treatment options for gastric cancer have been introduced but the prognosis of patients diagnosed with gastric cancer is still poor. Disease prognosis could be improved for high-risk individuals by implementing earlier screenings. Because many patients are asymptomatic during the early stages of gastric cancer, the diagnosis is often delayed and patients present with unresectable locally advanced or metastatic disease. Cytotoxic treatment has been shown to prolong survival in general, but not all patients are responders. The application of targeted therapies and multimodal treatment has improved prognosis for those with advanced disease. However, these new therapeutic strategies do not uniformly benefit all patients. Predicting whether patients will respond to specific therapies would be of particular value and would allow for stratifying patients for personalized treatment strategies. Metabolic imaging by positron emission tomography was the first technique with the potential to predict the response of esophago-gastric cancer to neoadjuvant therapy. Exploring and validating tissue-based biomarkers are ongoing processes. In this review, we discuss the status of several targeted therapies for gastric cancer, as well as proteomic and metabolic methods for investigating biomarkers for therapy response prediction in gastric cancer.
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Lai X, Schneider BP. Integrated and convenient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for LC-MS/MS analysis. Proteomics 2014; 14:2623-7. [DOI: 10.1002/pmic.201400110] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2014] [Revised: 07/09/2014] [Accepted: 07/31/2014] [Indexed: 11/10/2022]
Affiliation(s)
- Xianyin Lai
- Department of Biochemistry and Molecular Biology; Indiana University School of Medicine; Indianapolis IN USA
- Department of Cellular and Integrative Physiology; Indiana University School of Medicine; Indianapolis IN USA
| | - Bryan P. Schneider
- Department of Medicine; Indiana University School of Medicine; Indianapolis IN USA
- Department of Medical & Molecular Genetics; Indiana University School of Medicine; Indianapolis IN USA
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Mansour A, Chatila R, Bejjani N, Dagher C, Faour WH. A novel xylene-free deparaffinization method for the extraction of proteins from human derived formalin-fixed paraffin embedded (FFPE) archival tissue blocks. MethodsX 2014; 1:90-5. [PMID: 26150940 PMCID: PMC4472860 DOI: 10.1016/j.mex.2014.07.006] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2014] [Revised: 07/18/2014] [Accepted: 07/30/2014] [Indexed: 11/24/2022] Open
Abstract
Protein detection methods in formalin-fixed paraffin embedded (FFPE) tissue blocks are widely used in research and clinical setting in order to diagnose or to confirm a diagnosis of various types of diseases. Therefore, multiple protein extraction methods from FFPE tissue sections have been developed in this regard. However, the yield and the quality of proteins extracted from FFPE tissues are significantly reduced in blocks stored for longer periods of time. Regardless the protein extraction method used, tissue sections must be first deparaffinized with xylene, and then washed in serial dilutions of ethanol in order to remove the toxic organic solvent “xylene” and rehydrate the tissue. The objective of this study was first to develop a method to deparaffinize FFPE blocks that excludes the use of toxic solvent “xylene”. Second minimize the time required to perform the extraction. Here we describe a method where:
The entire paraffin embedded blocks are deparaffinized and rehydrated using only hot distilled water as a substitute for both xylene and ethanol The entire procedure takes about 15 min Deparaffinized blocks are immediately homogenized in lysis buffer, and the obtained lysate analyzed by Western blot. With this new modified technique, we were able to successfully detect actin and AKT proteins in lysates from blocks embedded in paraffin for up to 9 years.
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Affiliation(s)
- Anthony Mansour
- School of Medicine, Lebanese American University, Byblos, Lebanon
| | - Rajaa Chatila
- School of Medicine, Lebanese American University, Byblos, Lebanon ; Lebanese American University Medical Center - Rizk Hospital, Beirut, Lebanon
| | - Noha Bejjani
- Lebanese American University Medical Center - Rizk Hospital, Beirut, Lebanon
| | - Carole Dagher
- School of Medicine, Lebanese American University, Byblos, Lebanon
| | - Wissam H Faour
- School of Medicine, Lebanese American University, Byblos, Lebanon
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Tanca A, Abbondio M, Pisanu S, Pagnozzi D, Uzzau S, Addis MF. Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue. Clin Proteomics 2014; 11:28. [PMID: 25097466 PMCID: PMC4115481 DOI: 10.1186/1559-0275-11-28] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2014] [Accepted: 07/03/2014] [Indexed: 01/14/2023] Open
Abstract
BACKGROUND The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking. EXPERIMENTAL DESIGN DT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity. RESULTS DT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility. CONCLUSIONS These results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.
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Affiliation(s)
- Alessandro Tanca
- Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero, Italy
| | - Marcello Abbondio
- Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero, Italy
| | - Salvatore Pisanu
- Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero, Italy
| | - Daniela Pagnozzi
- Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero, Italy
| | - Sergio Uzzau
- Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero, Italy ; Dipartimento di Scienze Biomediche, Università di Sassari, Viale San Pietro 43/B, 07100, Sassari, Italy
| | - Maria Filippa Addis
- Porto Conte Ricerche, S.P. 55 Porto Conte/Capo Caccia Km 8.400, Tramariglio, 07041 Alghero, Italy
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