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Gobena S, Admassu B, Kinde MZ, Gessese AT. Proteomics and Its Current Application in Biomedical Area: Concise Review. ScientificWorldJournal 2024; 2024:4454744. [PMID: 38404932 PMCID: PMC10894052 DOI: 10.1155/2024/4454744] [Citation(s) in RCA: 13] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2023] [Revised: 02/09/2024] [Accepted: 02/13/2024] [Indexed: 02/27/2024] Open
Abstract
Biomedical researchers tirelessly seek cutting-edge technologies to advance disease diagnosis, drug discovery, and therapeutic interventions, all aimed at enhancing human and animal well-being. Within this realm, proteomics stands out as a pivotal technology, focusing on extensive studies of protein composition, structure, function, and interactions. Proteomics, with its subdivisions of expression, structural, and functional proteomics, plays a crucial role in unraveling the complexities of biological systems. Various sophisticated techniques are employed in proteomics, including polyacrylamide gel electrophoresis, mass spectrometry analysis, NMR spectroscopy, protein microarray, X-ray crystallography, and Edman sequencing. These methods collectively contribute to the comprehensive understanding of proteins and their roles in health and disease. In the biomedical field, proteomics finds widespread application in cancer research and diagnosis, stem cell studies, and the diagnosis and research of both infectious and noninfectious diseases. In addition, it plays a pivotal role in drug discovery and the emerging frontier of personalized medicine. The versatility of proteomics allows researchers to delve into the intricacies of molecular mechanisms, paving the way for innovative therapeutic approaches. As infectious and noninfectious diseases continue to emerge and the field of biomedical research expands, the significance of proteomics becomes increasingly evident. Keeping abreast of the latest developments in proteomics applications becomes paramount for the development of therapeutics, translational research, and study of diverse diseases. This review aims to provide a comprehensive overview of proteomics, offering a concise outline of its current applications in the biomedical domain. By doing so, it seeks to contribute to the understanding and advancement of proteomics, emphasizing its pivotal role in shaping the future of biomedical research and therapeutic interventions.
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Affiliation(s)
- Semira Gobena
- College of Veterinary Medicine and Animal Sciences, University of Gondar, Gondar, Ethiopia
| | - Bemrew Admassu
- Department of Veterinary Biomedical Sciences, College of Veterinary Medicine and Animal Sciences, University of Gondar, Gondar, Ethiopia
| | - Mebrie Zemene Kinde
- Department of Veterinary Biomedical Sciences, College of Veterinary Medicine and Animal Sciences, University of Gondar, Gondar, Ethiopia
| | - Abebe Tesfaye Gessese
- Department of Veterinary Biomedical Sciences, College of Veterinary Medicine and Animal Sciences, University of Gondar, Gondar, Ethiopia
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Al-Amrani S, Al-Jabri Z, Al-Zaabi A, Alshekaili J, Al-Khabori M. Proteomics: Concepts and applications in human medicine. World J Biol Chem 2021; 12:57-69. [PMID: 34630910 PMCID: PMC8473418 DOI: 10.4331/wjbc.v12.i5.57] [Citation(s) in RCA: 127] [Impact Index Per Article: 31.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/22/2021] [Revised: 05/04/2021] [Accepted: 07/15/2021] [Indexed: 02/06/2023] Open
Abstract
Proteomics is the complete evaluation of the function and structure of proteins to understand an organism’s nature. Mass spectrometry is an essential tool that is used for profiling proteins in the cell. However, biomarker discovery remains the major challenge of proteomics because of their complexity and dynamicity. Therefore, combining the proteomics approach with genomics and bioinformatics will provide an understanding of the information of biological systems and their disease alteration. However, most studies have investigated a small part of the proteins in the blood. This review highlights the types of proteomics, the available proteomic techniques, and their applications in different research fields.
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Affiliation(s)
- Safa Al-Amrani
- Department of Microbiology and Immunology, Sultan Qaboos University, Muscat 123, Oman
| | - Zaaima Al-Jabri
- Department of Microbiology and Immunology, Sultan Qaboos University, Muscat 123, Oman
| | - Adhari Al-Zaabi
- Department of Human and Clinical Anatomy, Sultan Qaboos University, Muscat 123, Oman
| | - Jalila Alshekaili
- Department of Microbiology and Immunology, Sultan Qaboos University Hospital, Muscat 123, Oman
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3
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Pouliquen DL, Boissard A, Coqueret O, Guette C. Biomarkers of tumor invasiveness in proteomics (Review). Int J Oncol 2020; 57:409-432. [PMID: 32468071 PMCID: PMC7307599 DOI: 10.3892/ijo.2020.5075] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2020] [Accepted: 05/07/2020] [Indexed: 12/13/2022] Open
Abstract
Over the past two decades, quantitative proteomics has emerged as an important tool for deciphering the complex molecular events involved in cancers. The number of references involving studies on the cancer metastatic process has doubled since 2010, while the last 5 years have seen the development of novel technologies combining deep proteome coverage capabilities with quantitative consistency and accuracy. To highlight key findings within this huge amount of information, the present review identified a list of tumor invasive biomarkers based on both the literature and data collected on a biocollection of experimental cell lines, tumor models of increasing invasiveness and tumor samples from patients with colorectal or breast cancer. Crossing these different data sources led to 76 proteins of interest out of 1,245 mentioned in the literature. Information on these proteins can potentially be translated into clinical prospects, since they represent potential targets for the development and evaluation of innovative therapies, alone or in combination. Herein, a systematical review of the biology of each of these proteins, including their specific subcellular/extracellular or multiple localizations is presented. Finally, as an important advantage of quantitative proteomics is the ability to provide data on all these molecules simultaneously in cell pellets, body fluids or paraffin‑embedded sections of tumors/invaded tissues, the significance of some of their interconnections is discussed.
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Affiliation(s)
| | - Alice Boissard
- Paul Papin ICO Cancer Center, CRCINA, Inserm, Université d'Angers, F‑44000 Nantes, France
| | | | - Catherine Guette
- Paul Papin ICO Cancer Center, CRCINA, Inserm, Université d'Angers, F‑44000 Nantes, France
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Chen L, Qin Y, Zhang T, Ding N, Chen Y, Zhang Z, Guo C. Clinical significance of cancer-associated fibroblasts and their correlation with microvessel and lymphatic vessel density in lung adenocarcinoma. J Clin Lab Anal 2019; 33:e22832. [PMID: 30737838 PMCID: PMC6528563 DOI: 10.1002/jcla.22832] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2018] [Revised: 10/24/2018] [Accepted: 11/19/2018] [Indexed: 12/21/2022] Open
Abstract
Background To determine whether cancer‐associated fibroblasts (CAFs) are associated with microvessel density (MVD) and lymphatic vessel density (LVD) in lung adenocarcinoma (ADC) or are not prognostic. Methods Ninety‐three lung adenocarcinoma patients without adjuvant therapy between January 2010 and June 2011 were enrolled. CAFs, MVD, and LVD were identified by α‐smooth muscle actin (α‐SMA), CD34 and D2‐40 staining via immunohistochemistry. Staining intensities were assessed and quantified. For statistics, Pearson's chi‐square test, logistic regression, Kaplan‐Meier, and log‐rank tests were applied. In addition, the Cox proportional hazards model was used for multifactor analysis to predict survival. Results CAFs abundance in lung adenocarcinoma is associated with higher MVD and LVD. In addition, a correlation was demonstrated between MVD and LVD (P < 0.05). CAFs, MVD, and LVD are significantly correlating with age, tumor size, differentiation grade, clinical stage, and lymph node metastasis (P < 0.05), but not influenced by gender, tumor location, and smoking history. Three‐year overall survival in the CAFs‐poor group is 64.5%, which is significant higher than that in the CAFs‐rich cohort (41.9%). Further, we found that age, clinical stage, α‐SMA, CD34, D2‐40 positivity, tumor size, differentiation grade, and lymph node metastasis significantly correlate with overall survival of patients with lung adenocarcinoma. However, sex, smoking history, and tumor location have no association with 3‐year survival. The clinical stage is an independent prognostic factor in overall survival (P < 0.05). Conclusions The density of CAFs identified by α‐SMA staining is associated with progression and metastasis of lung adenocarcinoma and affects the patient's disease outcome.
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Affiliation(s)
- Ling Chen
- Department of Oncology, Qingdao Municipal Hospital, Qingdao, China
| | - Yue Qin
- Department of Oncology, Qingdao Municipal Hospital, Qingdao, China
| | - Tenglong Zhang
- Department of Oncology, Qingdao Municipal Hospital, Qingdao, China
| | - Ning Ding
- Department of Oncology, University of Qingdao Medical School, Qingdao, China
| | - Yi Chen
- Department of Oncology, Qingdao Municipal Hospital, Qingdao, China
| | - Zhe Zhang
- Department of Thoracic Surgery, Qingdao Municipal Hospital, Qingdao, China
| | - Chengye Guo
- Department of Oncology, Qingdao Municipal Hospital, Qingdao, China
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Sharma S, Ray S, Moiyadi A, Sridhar E, Srivastava S. Quantitative proteomic analysis of meningiomas for the identification of surrogate protein markers. Sci Rep 2014; 4:7140. [PMID: 25413266 PMCID: PMC5382771 DOI: 10.1038/srep07140] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2014] [Accepted: 09/15/2014] [Indexed: 12/11/2022] Open
Abstract
Meningiomas are the most common non-glial tumors of the brain and spine. Pathophysiology and definite histological grading of meningiomas are frequently found to be deceptive due to their unusual morphological features and locations. Here for the first time we report a comprehensive serum proteomic analysis of different grades of meningiomas by using multiple quantitative proteomic and immunoassay-based approaches to obtain mechanistic insights about disease pathogenesis and identify grade specific protein signatures. In silico functional analysis revealed modulation of different vital physiological pathways including complement and coagulation cascades, metabolism of lipids and lipoproteins, immune signaling, cell growth and apoptosis and integrin signaling in meningiomas. ROC curve analysis demonstrated apolipoprotein E and A-I and hemopexin as efficient predictors for meningiomas. Identified proteins like vimentin, alpha-2-macroglobulin, apolipoprotein B and A-I and antithrombin-III, which exhibited a sequential increase in different malignancy grades of meningiomas, could serve as potential predictive markers.
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Affiliation(s)
- Samridhi Sharma
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
| | - Sandipan Ray
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
| | - Aliasgar Moiyadi
- Department of Neurosurgery, Advanced Center for Treatment Research and Education in Cancer, Tata Memorial Center, Kharghar, Navi Mumbai 410210, India
| | - Epari Sridhar
- Department of Pathology, Tata Memorial Hospital, Mumbai 400012, India
| | - Sanjeeva Srivastava
- Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
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Suganya N, Bhakkiyalakshmi E, Subin TS, Krishnamurthi K, Devi SS, Lau K, Sekar TV, Paulmurugan R, Ramkumar KM. Proteomic Identification of Pterostilbene-Mediated Anticancer Activities in HepG2 Cells. Chem Res Toxicol 2014; 27:1243-52. [DOI: 10.1021/tx5001392] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Affiliation(s)
- N. Suganya
- SRM
Research Institute, SRM University, Kattankulathur, Tamilnadu, India
| | - E. Bhakkiyalakshmi
- SRM
Research Institute, SRM University, Kattankulathur, Tamilnadu, India
| | - T. S. Subin
- Environmental
Health Division, National Environmental Engineering Research Institute, Nagpur, India
| | - K. Krishnamurthi
- Environmental
Health Division, National Environmental Engineering Research Institute, Nagpur, India
| | - S. Saravana Devi
- Environmental
Health Division, National Environmental Engineering Research Institute, Nagpur, India
| | - K. Lau
- Department
of Radiology, Stanford University School of Medicine, 3155 Porter
Drive, Stanford, California 94305, United States
| | - T. V. Sekar
- Department
of Radiology, Stanford University School of Medicine, 3155 Porter
Drive, Stanford, California 94305, United States
| | - R. Paulmurugan
- Department
of Radiology, Stanford University School of Medicine, 3155 Porter
Drive, Stanford, California 94305, United States
| | - K. M. Ramkumar
- SRM
Research Institute, SRM University, Kattankulathur, Tamilnadu, India
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Lin LL, Huang HC, Juan HF. Deciphering molecular determinants of chemotherapy in gastrointestinal malignancy using systems biology approaches. Drug Discov Today 2014; 19:1402-9. [PMID: 24793142 DOI: 10.1016/j.drudis.2014.04.016] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2014] [Revised: 03/15/2014] [Accepted: 04/24/2014] [Indexed: 12/26/2022]
Abstract
Gastrointestinal cancers are asymptomatic in early tumor development, leading to high mortality rates. Peri- or postoperative chemotherapy is a common strategy used to prolong the life expectancy of patients with these diseases. Understanding the molecular mechanisms by which anticancer drugs exert their effect is crucial to the development of anticancer therapies, especially when drug resistance occurs and an alternative drug is needed. By integrating high-throughput techniques and computational modeling to explore biological systems at different levels, from gene expressions to networks, systems biology approaches have been successfully applied in various fields of cancer research. In this review, we highlight chemotherapy studies that reveal potential signatures using microarray analysis, next-generation sequencing (NGS), proteomic and metabolomic approaches for the treatment of gastrointestinal cancers.
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Affiliation(s)
- Li-Ling Lin
- Institute of Molecular and Cellular Biology, Department of Life Science, National Taiwan University, Taipei, Taiwan
| | - Hsuan-Cheng Huang
- Institute of Biomedical Informatics, Center for Systems and Synthetic Biology, National Yang-Ming University, Taipei, Taiwan.
| | - Hsueh-Fen Juan
- Institute of Molecular and Cellular Biology, Department of Life Science, National Taiwan University, Taipei, Taiwan; Genome and Systems Biology Degree Program, National Taiwan University, Taipei, Taiwan; Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei, Taiwan.
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9
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Wienken U, Gaub HE. Stamping vital cells - a force-based ligand receptor assay. Biophys J 2013; 105:2687-94. [PMID: 24359740 PMCID: PMC3882508 DOI: 10.1016/j.bpj.2013.10.013] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2013] [Revised: 10/16/2013] [Accepted: 10/16/2013] [Indexed: 01/16/2023] Open
Abstract
Gaining information about receptor profiles on cells, and subsequently finding the most efficient ligands for these signaling receptors, remain challenging tasks in stem cell and cancer research as well as drug development. We introduce a live-cell method with great potential in both screening for surface receptors and analysing binding forces of different ligands. The technique is based on the molecular force assay, a parallel-format, high-throughput experiment on a single-molecule level. On human red blood cells, we demonstrate the detection of the interaction of N-acetyl-α-D-galactosaminyl residues with the lectin helix pomatia agglutinine and of the CD47 receptor with its antibody. The measurements are performed under nearly physiological conditions and still provide a highly specific binding signal. Moreover, with a detailed comparative force analysis on two cell types with different morphology, we show that our method even allows the determination of a DNA force equivalent for the interaction of the CD47 receptor and its antibody.
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Affiliation(s)
- Uta Wienken
- Chair of Experimental Physics & Center for NanoScience, Ludwig-Maximilians-University München, Munich, Germany
| | - Hermann E Gaub
- Chair of Experimental Physics & Center for NanoScience, Ludwig-Maximilians-University München, Munich, Germany.
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10
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Yang J, Liu P, Tian M, Li Y, Chen W, Li X. Proteomic identification of angiomotin by ProteomeLab PF-2D and correlation with clinical outcome in human clear cell renal cell carcinoma. Int J Oncol 2013; 42:2078-86. [PMID: 23588948 DOI: 10.3892/ijo.2013.1889] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2013] [Accepted: 03/13/2013] [Indexed: 11/05/2022] Open
Abstract
Identification of new therapeutic and prognostic biomarkers for clear cell renal cell carcinoma (ccRCC) is urgently required since most patients are in advanced stages of ccRCC at initial diagnosis and the recurrence rate is high. Differentially expressed proteins between the ccRCC cell line RLC-310 and the normal renal cell line HK-2 were identified by a comparative proteomic approach based on a protein fractionation two-dimensional (PF-2D) liquid-phase fractionation system and capillary liquid chromatography electrospray ionization mass spectrometry/mass spectrometry (LC-ESI-MS/MS). Differentially expressed proteins (n=196) were identified. Of the 13 differentially expressed proteins newly discovered in ccRCC, angiomotin (Amot) was the focus of this study since its role in ccRCC progression has been obscure. The overexpression of Amot in ccRCC tissues was confirmed by comparing Amot expression in 18 primary ccRCC tissues and adjacent normal renal tissues (ANRT) using western blot analysis. Quantitative RT-PCR using 127 ccRCC tissues revealed that high levels of Amot transcripts were associated with poor differentiation, venous invasion and decreased survival (p<0.0001, <0.05 and <0.05). Multivariate analysis indicated that Amot transcript was an independent prognostic factor for the survival of ccRCC patients (p<0.05). These data suggest that Amot may serve as a novel prognostic factor of ccRCC.
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Affiliation(s)
- Jin Yang
- Medical Oncology Department, The First Affiliated Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an 710061, P.R. China
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Rana S, Singla AK, Bajaj A, Elci SG, Miranda OR, Mout R, Yan B, Jirik FR, Rotello VM. Array-based sensing of metastatic cells and tissues using nanoparticle-fluorescent protein conjugates. ACS NANO 2012; 6:8233-40. [PMID: 22920837 PMCID: PMC3603354 DOI: 10.1021/nn302917e] [Citation(s) in RCA: 95] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/19/2023]
Abstract
Rapid and sensitive methods of discriminating between healthy tissue and metastases are critical for predicting disease course and designing therapeutic strategies. We report here the use of an array of gold nanoparticle-green fluorescent protein elements to rapidly detect metastatic cancer cells (in minutes), as well as to discriminate between organ-specific metastases and their corresponding normal tissues through their overall intracellular proteome signatures. Metastases established in a new preclinical non-small-cell lung cancer metastasis model in athymic mice were used to provide a challenging and realistic testbed for clinical cancer diagnosis. Full differentiation between the analyte cell/tissue was achieved with as little as 200 ng of intracellular protein (~1000 cells) for each nanoparticle, indicating high sensitivity of this sensor array. Notably, the sensor created a distinct fingerprint pattern for the normal and metastatic tumor tissues. Moreover, this array-based approach is unbiased, precluding the requirement of a priori knowledge of the disease biomarkers. Taken together, these studies demonstrate the utility of this sensor for creating fingerprints of cells and tissues in different states and present a generalizable platform for rapid screening amenable to microbiopsy samples.
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Affiliation(s)
- Subinoy Rana
- Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, USA
| | - Arvind K. Singla
- Department of Biochemistry and Molecular Biology, The McCaig Institute for Bone and Joint Health, University of Calgary, Calgary, Alberta, Canada
| | - Avinash Bajaj
- Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, USA
| | - S. Gokhan Elci
- Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, USA
| | - Oscar R. Miranda
- Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, USA
| | - Rubul Mout
- Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, USA
| | - Bo Yan
- Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, USA
| | - Frank R. Jirik
- Department of Biochemistry and Molecular Biology, The McCaig Institute for Bone and Joint Health, University of Calgary, Calgary, Alberta, Canada
| | - Vincent M. Rotello
- Department of Chemistry, University of Massachusetts, Amherst, Massachusetts, USA
- Corresponding Author: Vincent M. Rotello;
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Pietsch J, Sickmann A, Weber G, Bauer J, Egli M, Wildgruber R, Infanger M, Grimm D. Metabolic enzyme diversity in different human thyroid cell lines and their sensitivity to gravitational forces. Proteomics 2012; 12:2539-46. [PMID: 22707460 DOI: 10.1002/pmic.201200070] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2012] [Revised: 04/20/2012] [Accepted: 05/15/2012] [Indexed: 12/25/2022]
Abstract
Many cancer cells show unique protein expression patterns. We used proteome technology including MS, free flow isoelectric focusing and Western blotting to determine current concentrations of metabolic enzymes in healthy and malignant human thyroid cells. Three different types of human thyroid cells were investigated after they had been cultured under equal conditions. MS revealed high quantities of glycolytic enzymes and moderate quantities of citric acid cycle enzymes in malignant FTC-133 cells with abnormal LDH B-chains, high quantities of glycolytic enzymes and marginal quantities of citric acid cycle enzymes in normal HTU-5 cells, and low quantities of glycolytic enzymes and marginal quantities of citrate cycle enzymes in malignant CGTH-W1 cells with abnormal LDH A-chains. When an alteration of gene expression activity was challenged physically by removing gravity forces, the concentrations of various glycolytic enzymes were changed in normal and malignant thyroid cells. However, the changes varied among the different cell types. Different cellular alignment of the enzymes could be one reason for the cell type-specific behavior as demonstrated by histological analysis of alpha-enolase.
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Affiliation(s)
- Jessica Pietsch
- Plastic, Aesthetic and Hand Surgery, Otte-von Guericke University, Magdeburg, Germany
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13
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Choi JW, Liu H, Song H, Park JHY, Yun JW. Plasma marker proteins associated with the progression of lung cancer in obese mice fed a high-fat diet. Proteomics 2012; 12:1999-2013. [DOI: 10.1002/pmic.201100582] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Affiliation(s)
- Jung-Won Choi
- Department of Biotechnology,; Daegu University,; Kyungsan; Kyungbuk; Republic of Korea
| | - Hao Liu
- Department of Biotechnology,; Daegu University,; Kyungsan; Kyungbuk; Republic of Korea
| | - Hyerim Song
- Department of Food Science and Nutrition; Hallym University; Chuncheon; Republic of Korea
| | - Jung Han Yoon Park
- Department of Food Science and Nutrition; Hallym University; Chuncheon; Republic of Korea
| | - Jong Won Yun
- Department of Biotechnology,; Daegu University,; Kyungsan; Kyungbuk; Republic of Korea
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Proteomics in melanoma biomarker discovery: great potential, many obstacles. INTERNATIONAL JOURNAL OF PROTEOMICS 2011; 2011:181890. [PMID: 22084682 PMCID: PMC3195774 DOI: 10.1155/2011/181890] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 07/12/2011] [Accepted: 08/02/2011] [Indexed: 01/22/2023]
Abstract
The present clinical staging of melanoma stratifies patients into heterogeneous groups, resulting in the application of aggressive therapies to large populations, diluting impact and increasing toxicity. To move to a new era of therapeutic decisions based on highly specific tumor profiling, the discovery and validation of new prognostic and predictive biomarkers in melanoma is critical. Genomic profiling, which is showing promise in other solid tumors, requires fresh tissue from a large number of primary tumors, and thus faces a unique challenge in melanoma. For this and other reasons, proteomics appears to be an ideal choice for the discovery of new melanoma biomarkers. Several approaches to proteomics have been utilized in the search for clinically relevant biomarkers, but to date the results have been relatively limited. This article will review the present work using both tissue and serum proteomics in the search for melanoma biomarkers, highlighting both the relative advantages and disadvantages of each approach. In addition, we review several of the major obstacles that need to be overcome in order to advance the field.
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Durbin KR, Tran JC, Zamdborg L, Sweet SMM, Catherman AD, Lee JE, Li M, Kellie JF, Kelleher NL. Intact mass detection, interpretation, and visualization to automate Top-Down proteomics on a large scale. Proteomics 2011; 10:3589-97. [PMID: 20848673 DOI: 10.1002/pmic.201000177] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Applying high-throughput Top-Down MS to an entire proteome requires a yet-to-be-established model for data processing. Since Top-Down is becoming possible on a large scale, we report our latest software pipeline dedicated to capturing the full value of intact protein data in automated fashion. For intact mass detection, we combine algorithms for processing MS1 data from both isotopically resolved (FT) and charge-state resolved (ion trap) LC-MS data, which are then linked to their fragment ions for database searching using ProSight. Automated determination of human keratin and tubulin isoforms is one result. Optimized for the intricacies of whole proteins, new software modules visualize proteome-scale data based on the LC retention time and intensity of intact masses and enable selective detection of PTMs to automatically screen for acetylation, phosphorylation, and methylation. Software functionality was demonstrated using comparative LC-MS data from yeast strains in addition to human cells undergoing chemical stress. We further these advances as a key aspect of realizing Top-Down MS on a proteomic scale.
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Affiliation(s)
- Kenneth R Durbin
- Department of Chemistry, The Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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Ortiz S, Lee W, Smith D, Forman SJ, Lee TD, Liu CP. Comparative analyses of differentially induced T-cell receptor-mediated phosphorylation pathways in T lymphoma cells. Exp Biol Med (Maywood) 2010; 235:1450-63. [PMID: 21127342 PMCID: PMC3247199 DOI: 10.1258/ebm.2010.010056] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Activation of T lymphoma cells expressing Syk, but not ZAP-70 tyrosine kinase, has been shown to negatively regulate cell activation and activation-induced cell death (AICD), perhaps due to differential induction of tyrosine phosphorylation modified proteins. To better understand the role of these proteins and their associated molecules/pathways, we studied a previously described model of T lymphoma cells expressing either a kinase-activated chimeric Syk or ZAP-70 genetically linked to T-cell receptor (TCR) ζ chain (Z/Syk or Z/ZAP cells, respectively). To help identify molecules and pathways linked to cell activation or AICD, a comparative semi-quantitative proteomics-based approach was utilized to analyze tyrosine-phosphorylated protein immunoprecipitates from two-minute short-term activated Z/Syk or Z/ZAP cells. Using the resulting bioinformatics data-sets, we identified several differentially immunoprecipitated proteins that could be validated biochemically. More tyrosine-phosphorylated and phosphotyrosine-associated proteins were found in Z/Syk than in Z/ZAP cells. Proteins involved in different unique functional pathways were induced in these cells and showed altered intermolecular interactions in varied pathways. Remarkably, 41% of differentially identified proteins in Z/Syk cells belonged to cell cycle or vesicle/trafficking pathways. In contrast, 21% of such proteins in Z/ZAP cells belonged to metabolism pathways. Therefore, molecular pathways involved in post-translational modifications linked to distinct cellular/physiological functions are differentially activated, which may contribute to varied activation and AICD responses of these cells. In summary, we identified proteins belonging to novel differentially activated pathways involved in TCR-mediated signaling, which may be targets for regulating activation and AICD of T lymphoma cells and for potential cancer therapy.
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Affiliation(s)
- Serina Ortiz
- Department of Immunology, Beckman Research Institute, City of Hope 1450 E. Duarte Rd., Duarte, CA 91010-3000
| | - Wenhui Lee
- Department of Immunology, Beckman Research Institute, City of Hope 1450 E. Duarte Rd., Duarte, CA 91010-3000
| | - David Smith
- Department of Information Sciences, Beckman Research Institute, City of Hope 1450 E. Duarte Rd., Duarte, CA 91010-3000
| | - Stephen J. Forman
- Department of Hematology, Beckman Research Institute, City of Hope 1450 E. Duarte Rd., Duarte, CA 91010-3000
| | - Terry D. Lee
- Department of Immunology, Beckman Research Institute, City of Hope 1450 E. Duarte Rd., Duarte, CA 91010-3000
| | - Chih-Pin Liu
- Department of Immunology, Beckman Research Institute, City of Hope 1450 E. Duarte Rd., Duarte, CA 91010-3000
- Department of Diabetes and Metabolism Research, Beckman Research Institute, City of Hope 1450 E. Duarte Rd., Duarte, CA 91010-3000
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Moritz RL, Skandarajah AR, Ji H, Simpson RJ. Proteomic analysis of colorectal cancer: prefractionation strategies using two-dimensional free-flow electrophoresis. Comp Funct Genomics 2010; 6:236-43. [PMID: 18629191 PMCID: PMC2447484 DOI: 10.1002/cfg.477] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2005] [Revised: 03/16/2005] [Accepted: 03/17/2005] [Indexed: 01/21/2023] Open
Abstract
This review deals with the application of a new prefractionation tool, free-flow
electrophoresis (FFE), for proteomic analysis of colorectal cancer (CRC). CRC is a
leading cause of cancer death in the Western world. Early detection is the single most
important factor influencing outcome of CRC patients. If identified while the disease
is still localized, CRC is treatable. To improve outcomes for CRC patients there
is a pressing need to identify biomarkers for early detection (diagnostic markers),
prognosis (prognostic indicators), tumour responses (predictive markers) and disease
recurrence (monitoring markers). Despite recent advances in the use of genomic
analysis for risk assessment, in the area of biomarker identification genomic methods
alone have yet to produce reliable candidate markers for CRC. For this reason,
attention is being directed towards proteomics as a complementary analytical tool
for biomarker identification. Here we describe a proteomics separation tool, which
uses a combination of continuous FFE, a liquid-based isoelectric focusing technique, in
the first dimension, followed by rapid reversed-phase HPLC (1–6 min/analysis) in the
second dimension. We have optimized imaging software to present the FFE/RP-HPLC
data in a virtual 2D gel-like format. The advantage of this liquid based fractionation
system over traditional gel-based fractionation systems is the ability to fractionate
large quantity protein samples. Unlike 2D gels, the method is applicable to both
high-Mr proteins and small peptides, which are difficult to separate, and in the case
of peptides, are not retained in standard 2D gels.
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Affiliation(s)
- Robert L Moritz
- Joint Proteomics Laboratory Ludwig Institute for Cancer Research (Melbourne Branch), The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia
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Mathias RA, Chen YS, Wang B, Ji H, Kapp EA, Moritz RL, Zhu HJ, Simpson RJ. Extracellular remodelling during oncogenic Ras-induced epithelial-mesenchymal transition facilitates MDCK cell migration. J Proteome Res 2010; 9:1007-19. [PMID: 19954229 DOI: 10.1021/pr900907g] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Epithelial-mesenchymal transition (EMT) describes a process whereby immotile epithelial cells escape structural constraints imposed by cellular architecture and acquire a phenotype characteristic of migratory mesenchymal cells. Implicated in carcinoma progression and metastasis, EMT has been the focus of several recent proteomics-based studies aimed at identifying new molecular players. To gain insights into extracellular mediators associated with EMT, we conducted an extensive proteomic analysis of the secretome from MDCK cells following oncogenic Ras-induced EMT (21D1 cells). Using Orbitrap technology and a label-free quantitative approach, differential expression of several secreted modulators were revealed. Proteomic findings were further substantiated by mRNA transcript expression analysis with 71% concordance. MDCK cells undergoing Ras-induced EMT remodel the extracellular matrix (ECM) via diminished expression of basement membrane constituents (collagen type IV and laminin 5), up-regulation of extracellular proteases (MMP-1, kallikreins -6 and -7), and increased production and secretion of ECM constituents (SPARC, collagen type I, fibulins -1 and -3, biglycan, and decorin). Collectively, these findings suggest that hierarchical regulation of a subset of extracellular effectors may coordinate a biological response during EMT that enhances cell motility. Transient silencing of MMP-1 in 21D1 cells via siRNA-mediated knockdown attenuated cell migration. Many of the secretome proteins identified broaden our understanding of the EMT process.
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Affiliation(s)
- Rommel A Mathias
- Joint Proteomics Laboratory, Ludwig Institute for Cancer Research, Parkville, Victoria, Australia
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19
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Saydam O, Senol O, Schaaij-Visser TBM, Pham TV, Piersma SR, Stemmer-Rachamimov AO, Wurdinger T, Peerdeman SM, Jimenez CR. Comparative protein profiling reveals minichromosome maintenance (MCM) proteins as novel potential tumor markers for meningiomas. J Proteome Res 2010; 9:485-94. [PMID: 19877719 DOI: 10.1021/pr900834h] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Meningiomas are among the most frequent tumors of the brain and spinal cord accounting for 15-20% of all central nervous system tumors and frequently associated with neurofibromatosis type 2. In this study, we aimed to unravel molecular meningioma tumorigenesis and discover novel protein biomarkers for diagnostic and/or prognostic purposes and performed in-depth proteomic profiling of meningioma cells compared to human primary arachnoidal cells. We isolated proteins from meningioma cell line SF4433 and human primary arachnoidal cells and analyzed the protein profiles by Gel-nanoLC-MS/MS in conjunction with protein identification and quantification by shotgun nanoLC tandem mass spectrometry and spectral counting. Differential analysis of meningiomas revealed changes in the expression levels of 281 proteins (P < 0.01) associated with various biological functions such as DNA replication, recombination, cell cycle, and apoptosis. Among several interesting proteins, we focused on a subset of the highly significantly up-regulated proteins, the minichromosome maintenance (MCM) family. We performed subsequent validation studies by qRT-PCR in human meningioma tissue samples (WHO grade I, 14 samples; WHO grade II, 7 samples; and WHO grade III, 7 samples) compared to arachnoidal tissue controls (from fresh autopsies; 3 samples) and found that MCMs are highly and significantly up-regulated in human meningioma tumor samples compared to arachnoidal tissue controls. We found a significant increase in MCM2 (8 fold), MCM3 (5 fold), MCM4 (4 fold), MCM5 (4 fold), MCM6 (3 fold), and MCM7 (5 fold) expressions in meningiomas. This study suggests that MCM family proteins are up-regulated in meningiomas and can be used as diagnostic markers.
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Affiliation(s)
- Okay Saydam
- Department of Neurology and Radiology, Massachusetts General Hospital, and Neuroscience Program, Harvard Medical School, Boston, Massachusetts, 02129, USA.
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20
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Cutillas PR, Timms JF. Approaches and applications of quantitative LC-MS for proteomics and activitomics. Methods Mol Biol 2010; 658:3-17. [PMID: 20839095 DOI: 10.1007/978-1-60761-780-8_1] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/29/2023]
Abstract
LC-MS is a powerful technique in biomolecular research. In addition to its uses as a tool for protein and peptide quantization, LC-MS can also be used to quantify the activity of signalling and metabolic pathways in a multiplex and comprehensive manner, i.e. as an 'activitomic' tool. Taking cancer research as an illustrative example of application, this review discusses the concepts of biochemical pathway analysis using LC-MS-based proteomic and activitomic techniques.
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Affiliation(s)
- Pedro R Cutillas
- Analytical Signalling Group, Centre for Cell Signalling, Institute of Cancer, Bart's and the London School of Medicine, Queen Mary University of London, London, UK
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21
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Kim HH, Kim T, Kim E, Park JK, Park SJ, Joo H, Kim HJ. The Mitochondrial Warburg Effect: A Cancer Enigma. ACTA ACUST UNITED AC 2009. [DOI: 10.4051/ibc.2009.2.0007] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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22
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Chung JC, Oh MJ, Choi SH, Bae CD. Proteomic analysis to identify biomarker proteins in pancreatic ductal adenocarcinoma. ANZ J Surg 2008; 78:245-51. [PMID: 18366394 DOI: 10.1111/j.1445-2197.2008.04429.x] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
BACKGROUND Pancreatic ductal adenocarcinoma (PDAC) is the fifth most common cause of death from cancer in Korea. PDAC is difficult to diagnose at an early stage and even more difficult to cure. Thus, there is an urgent need to identify molecular targets for early diagnosis and effective treatment. The objectives of this study were to identify differentially expressed biomarker proteins of PDAC using proteomic analysis, to validate the identified biomarker proteins associated with carcinogenesis using western blot analysis and to evaluate clinical factors influencing expression of candidate biomarker proteins. METHODS In the present study, we carried out proteomic analysis in 10 pairs of PDAC specimens with matching adjacent normal tissues to clarify the different patterns of protein expression. The proteins were separated by high-resolution 2-D polyacrylamide gel electrophoresis (2D PAGE) and the differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Differential expression of candidate biomarker proteins associated with carcinogenesis was further validated using western blot analysis. Standard statistical analysis was carried out in an attempt to establish a correlation between clinical variables and expression of candidate biomarker proteins. RESULTS Analysis of PDAC and the adjacent normal tissues showed reproducibly similar proteomic patterns for each group. Approximately 700 spots each were seen by silver-stained gels from both PDAC and normal tissues. Differentially expressed protein spots were gel digested and identified by MALDI-TOF MS. Twenty-five proteins were identified, of which five proteins (galectin-1, enolase-2, alpha-1-antitrypsin, N-myc interactor, peroxiredoxin-4) were previously reported as being differentially expressed either at the mRNA level or protein level in human cancer. The five proteins were selected for candidate biomarker proteins related to carcinogenesis. These proteins were further validated by western blot analysis. Among the candidate biomarker proteins, galectin-1 expression was highly correlated to histology (P = 0.019), T stage (P = 0.047), N stage (P = 0.033) and American Joint Committee on Cancer stage (P = 0.011). CONCLUSION Differentially expressed 25 proteins in PDAC were identified using proteomic analysis and five proteins related to carcinogenesis were validated by western blot analysis. Galectin-1 expression was highly correlated to tumour histology and stage.
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Affiliation(s)
- Jun Chul Chung
- Department of Surgery, Soonchunhyang University College of Medicine, Soonchunhyang University Bucheon Hospital, Bucheon, Korea
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23
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Streckfus CF, Mayorga-Wark O, Arreola D, Edwards C, Bigler L, Dubinsky WP. Breast cancer related proteins are present in saliva and are modulated secondary to ductal carcinoma in situ of the breast. Cancer Invest 2008; 26:159-67. [PMID: 18259946 DOI: 10.1080/07357900701783883] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
OBJECTIVE The objective of this study was to determine if protein-by-products secondary to cancer related oncogenes appear in the saliva of breast cancer patients. METHODS Three pooled (n = 10 subjects/pool) stimulated whole saliva specimens from women were analyzed. One pooled specimen was from healthy women, another pooled specimen from women diagnosed with a benign breast tumor and the other one pooled specimen was from women diagnosed with ductal carcinoma in situ (DCIS). Differential expression of proteins was measured by isotopically tagging proteins in the tumor groups and comparing them to the healthy control group. Experimentally, saliva from each of the pooled samples was trypsinized and the peptide digests labeled with the appropriate iTRAQ reagent. Labeled peptides from each of the digests were combined and analyzed by reverse phase (C18) capillary chromatography on an Applied Biosystems QStar LC-MS/MS mass spectrometer equipped with an LC-Packings HPLC. RESULTS The results of the salivary analyses in this population of patients yielded approximately 130 proteins in the saliva specimens. Forty-nine proteins were differentially expressed between the healthy control pool and the benign and cancer patient groups. CONCLUSIONS The study suggests that saliva is a fluid suffused with solubilized by-products of oncogenic expression and that these proteins may be modulated secondary to DCIS. Additionally, there may be salivary protein profiles that are unique to both DCIS and fibroadenoma tumors.
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Affiliation(s)
- Charles F Streckfus
- University of Texas Health Science Center-Dental Branch, Houston, Texas 77030, USA.
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Bitarte N, Bandrés E, Zárate R, Ramirez N, Garcia-Foncillas J. Moving forward in colorectal cancer research, what proteomics has to tell. World J Gastroenterol 2007; 13:5813-21. [PMID: 17990347 PMCID: PMC4205428 DOI: 10.3748/wjg.v13.i44.5813] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Colorectal cancer is the third most common cancer and is highly fatal. During the last several years, research has been primarily based on the study of expression profiles using microarray technology. But now, investigators are putting into practice proteomic analyses of cancer tissues and cells to identify new diagnostic or therapeutic biomarkers for this cancer. Because the proteome reflects the state of a cell, tissue or organism more accurately, much is expected from proteomics to yield better tumor markers for disease diagnosis and therapy monitoring. This review summarizes the most relevant applications of proteomics the biomarker discovery for colorectal cancer.
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Pardo M, Dwek RA, Zitzmann N. Proteomics in uveal melanoma research: opportunities and challenges in biomarker discovery. Expert Rev Proteomics 2007; 4:273-86. [PMID: 17425462 DOI: 10.1586/14789450.4.2.273] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Uveal melanoma (UM) is the most frequent primary intraocular tumor in adult humans. Despite the significant advances in diagnosis and treatment of UM in the last decades, the prognosis of UM sufferers is still poor. Metastatic liver disease is the leading cause of death in UM and can develop after a long disease-free interval, suggesting the presence of occult micrometastasis. Proteomics technology has opened new opportunities for elucidating the molecular mechanism of complex diseases, such as cancer. This article will review the recent developments in biomarker discovery for UM research by proteomics. In the last few years, the first UM proteomics-based analyses have been launched, yielding promising results. An update on recent developments on this field is presented.
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Affiliation(s)
- María Pardo
- Universidad de Santiago de Compostela, Laboratorio de Endocrinología Molecular, Departamento de Medicina, Complejo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain.
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26
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Mathelin C, Cromer A, Wendling C, Tomasetto C, Rio MC. Serum biomarkers for detection of breast cancers: A prospective study. Breast Cancer Res Treat 2007; 96:83-90. [PMID: 16322896 DOI: 10.1007/s10549-005-9046-2] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
Using surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF), Li et al. [Clin Chem 48(8): 1296-1304, 2002] identified 3 serum biomarkers, BC1 (4.3 kDa), BC2 (8.1 kDa) and BC3 (8.9 kDa), whose combination significantly detects breast cancer patients from non-cancer controls. This work aimed to validate these biomarkers in an independent prospective study. We screened 89 serum samples including 49 breast cancers at pT1-4N0M0 (n = 23), pT1-4N1-3M0 (n = 17) or pT1-4N0-3M1 (n = 9) stages, 13 benign breast diseases and 27 healthy women. The BC2 biomarker significance was not recovered. However, we found 2 peaks that we named BC1a (4286 Da) and BC1b (4302 Da), that could correspond to Li's BC1 since they significantly decrease in breast cancers (p < 0.00007 and p < 0.0002, respectively). Similarly, BC3a (8919 Da) and BC3b (8961 Da) are significantly increased in breast cancers (p < 0.02 and p < 0.0002, respectively) and could correspond to the Li's BC3. For each biomarker we defined stringent (no errors) and flexible (less than 10% errors) cut-off values and tested the power of the combined BC1a/BC1b/BC3a/BC3b stringent and flexible profiles to discriminate breast cancers. They identified 33% and 45% cancers, respectively. Applied to the same series, Ca 15.3 test identified 22% patients. Interestingly, in association with the BC1a/BC1b/BC3a/BC3b profiles, Ca 15.3 improved the number of detected cancers indicating that it is an independent parameter. Collectively, our data partially validate those of Li's study and confirm that the BC1 and BC3 biomarkers are helpful for breast cancer diagnosis.
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MESH Headings
- Adenocarcinoma, Mucinous/blood
- Adenocarcinoma, Mucinous/diagnosis
- Adult
- Aged
- Aged, 80 and over
- Biomarkers, Tumor/blood
- Breast Neoplasms/blood
- Breast Neoplasms/diagnosis
- Carcinoma, Ductal, Breast/blood
- Carcinoma, Ductal, Breast/diagnosis
- Carcinoma, Lobular/blood
- Carcinoma, Lobular/diagnosis
- Female
- Humans
- Middle Aged
- Prognosis
- Prospective Studies
- Proteomics
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Affiliation(s)
- Carole Mathelin
- Hôpitaux Universitaires de Strasbourg, 1, place de l'hôpital, Strasbourg Cedex, France
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Park YM, Kim JY, Kwon KH, Lee SK, Kim YH, Kim SY, Park GW, Lee JH, Lee B, Yoo JS. Profiling human brain proteome by multi-dimensional separations coupled with MS. Proteomics 2006; 6:4978-86. [PMID: 16927429 DOI: 10.1002/pmic.200600098] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
In our initial attempt to analyze the human brain proteome, we applied multi-dimensional protein separation and identification techniques using a combination of sample fractionation, 1-D SDS-PAGE, and MS analysis. The complexity of human brain proteome requires multiple fractionation strategies to extend the range and total number of proteins identified. According to the method of Klose (Methods Mol. Biol. 1999, 112, 67), proteins of the temporal lobe of human brain were fractionated into (i) cytoplasmic and nucleoplasmic, (ii) membrane and other structural, and (iii) DNA-binding proteins. Each fraction was then separated by SDS-PAGE, and the resulting gel line was cut into approximately 50 bands. After trypsin digestion, the resulting peptides from each band were analyzed by RP-LC/ESI-MS/MS using an LTQ spectrometer. The SEQUEST search program, which searched against the IPI database, was used for peptide sequence identification, and peptide sequences were validated by reversed sequence database search and filtered by the Protein Hit Score. Ultimately, 1533 proteins could be detected from the human brain. We classified the identified proteins according to their distribution on cellular components. Among these proteins, 24% were membrane proteins. Our results show that the multiple separation strategy is effective for high-throughput characterization of proteins from complex proteomic mixtures.
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Affiliation(s)
- Young Mok Park
- Proteomics Team, Korea Basic Science Institute, Daejeon, Republic of Korea
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28
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Stem cells and proteomics. Chin J Cancer Res 2006. [DOI: 10.1007/s11670-006-0161-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
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Hwang TL, Liang Y, Chien KY, Yu JS. Overexpression and elevated serum levels of phosphoglycerate kinase 1 in pancreatic ductal adenocarcinoma. Proteomics 2006; 6:2259-72. [PMID: 16493704 DOI: 10.1002/pmic.200500345] [Citation(s) in RCA: 105] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a common malignancy with a very low 5-year survival rate. Currently, there are no valid markers for early detection and targets for therapy. Here, we used 2-DE to analyze the protein profiles of eight PDAC specimens and paired adjacent nontumor tissues. MS was used to identify 15 protein spots that were found to be overexpressed in PDAC tissues versus adjacent controls. One of them was identified as phosphoglycerate kinase (PGK) 1, a secretable glycolytic enzyme known to participate in angiogenesis. Immunohistochemical analysis of 63 PDAC specimens revealed moderate to strong expression of PGK1 in >70% of the tumors. Further Western blotting analysis of cells from tumor and adjacent nontumor tissues obtained by laser capture microdissection confirmed the enhanced expression of PGK1 in tumor cells. Furthermore, the serum levels of PGK1 were significantly higher in PDAC patients (n = 21) than in the control group (n = 25) (p < 0.005), as determined by ELISA. These observations indicate that protein profile analysis using a combination of 2-DE and MS provides an effective strategy for identifying biomarkers that may have diagnostic potential for PDAC, and identify PGK1 as a potential biomarker and/or therapeutic target for PDAC.
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Affiliation(s)
- Tsann-Long Hwang
- Department of Surgery, Chang Gung Memorial Hospital, Chang Gung University, Tao-Yuan, Taiwan
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Carta F, Demuro PP, Zanini C, Santona A, Castiglia D, D'Atri S, Ascierto PA, Napolitano M, Cossu A, Tadolini B, Turrini F, Manca A, Sini MC, Palmieri G, Rozzo AC. Analysis of candidate genes through a proteomics-based approach in primary cell lines from malignant melanomas and their metastases. Melanoma Res 2006; 15:235-44. [PMID: 16034300 DOI: 10.1097/00008390-200508000-00002] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
Proteomics provides a powerful approach for screening alterations in protein expression and post-translational modification associated with particular human diseases. In this study, the analysis of protein expression was focused on malignant melanoma in order to determine the candidate genes involved in tumour progression. The proteomes of cultured melanocytes and of cell lines from primary and metastatic lesions of one malignant melanoma patient were profiled using two-dimensional electrophoresis (2-DE) and mass spectrometry. Differentially expressed proteins were confirmed by 2-DE and mass spectrometry on an additional four malignant melanoma cell lines. Total RNA from the first subset of cell lines was used for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of the candidate genes identified after proteomics analysis. A very high similarity was observed in the 2-DE maps of two malignant melanoma cell lines derived from primary and secondary lesions of the same patient. Mass spectrometry identified 37 proteins which were found to be more abundant in tumour cells in comparison with control melanocytes (as confirmed on additional cell lines), with a relatively high prevalence of stress proteins. Eight candidate genes (PRDX2, HSP27, HSP60, HSPA8, HSP9B, STIP1, PDI and P4HB) were further characterized by evaluating their messenger RNA expression levels through real-time RT-PCR analysis. Overexpression of HSP27, HSP60 and HSPA8 and downregulation of PRDX2 were observed in cells from metastatic malignant melanoma in comparison with those from primary melanoma. Although further investigations with larger numbers of paired normal and tumour samples are needed, our findings strongly suggest that the dysregulation of stress pathways may be involved in melanoma progression.
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Papassotiropoulos A, Fountoulakis M, Dunckley T, Stephan DA, Reiman EM. Genetics, transcriptomics, and proteomics of Alzheimer's disease. J Clin Psychiatry 2006; 67:652-70. [PMID: 16669732 PMCID: PMC2259384 DOI: 10.4088/jcp.v67n0418] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
OBJECTIVE To provide an updated overview of the methods used in genetic, transcriptomic, and proteomic studies in Alzheimer's disease and to demonstrate the importance of those methods for the improvement of the current diagnostic and therapeutic possibilities. DATA SOURCES MEDLINE-based search of 233 peer-reviewed articles published between 1975 and 2006. DATA SYNTHESIS Alzheimer's disease is a genetically heterogeneous disorder. Rare mutations in the amyloid precursor protein, presenilin 1, and presenilin 2 genes have shown the importance of the amyloid metabolism for its development. In addition, converging evidence from population-based genetic studies, gene expression studies, and protein profile studies in the brain and in the cerebrospinal fluid suggest the existence of several pathogenetic pathways such as amyloid precursor protein processing, beta-amyloid degradation, tau phosphorylation, proteolysis, protein misfolding, neuroinflammation, oxidative stress, and lipid metabolism. CONCLUSIONS The development of high-throughput genotyping methods and of elaborated statistical analyses will contribute to the identification of genetic risk profiles related to the development and course of this devastating disease. The integration of knowledge derived from genetic, transcriptomic, and proteomic studies will greatly advance our understanding of the causes of Alzheimer's disease, improve our capability of establishing an early diagnosis, help define disease subgroups, and ultimately help to pave the road toward improved and tailored treatments.
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Mojica WD, Rapkiewicz AV, Liotta LA, Espina V. Manual exfoliation of fresh tissue obviates the need for frozen sections for molecular profiling. Cancer 2006; 105:483-91. [PMID: 16015639 DOI: 10.1002/cncr.21347] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
BACKGROUND Simple, rapid tissue processing that preserves macromolecules will enhance translational research capabilities. Traditional fixative-based approaches for specimen preservation are ideal for histologic evaluation but are not conducive to molecular studies of nucleic acids and protein. Tissue cryosections preserve macromolecule integrity, but the process is labor intensive and technically challenging. To the authors' knowledge to date, an alternative method capable of retrieving cells while providing adequate histologic detail yet preserving macromolecule integrity has been lacking. In the current study, the authors evaluated the utility of using manual exfoliation of clinical tissue samples as a means of obtaining cells for molecular analysis. This technique possesses the advantages of fixed and frozen tissue sections without their drawbacks. This simple, rapid, nonfixative based technique is capable of preparing cells from human clinical material for further isolation without compromising the preservation of macromolecules in the tissue. METHODS Cells from a variety of clinical resection specimens from solid tumors were directly scraped from the tissue samples using the edge of a glass microscope slide and smeared onto another slide for cytologic evaluation. The manually exfoliated cells were evaluated microscopically for cytologic quality and cellular quantity. Pure cell populations were procured by laser capture microdissection (LCM) with subsequent extraction of nucleic acids and proteins. The integrity and suitability of the recovered nucleic acids and proteins for molecular analysis were evaluated using the polymerase chain reaction (PCR), reverse transcriptase-PCR, and reverse-phase protein microarray, respectively. RESULTS Manual exfoliation permits the selection of homogeneous cell populations by LCM based on well established cytologic characteristics. DNA and mRNA, of comparable quality to frozen sections, can be amplified from the manual exfoliation cells. Proteins of similar quality can be recovered using this technique and quantitated via reverse-phase protein microarray. CONCLUSIONS Molecular macromolecules of high quality and sufficient quantity can be retrieved from human clinical samples using manual exfoliation and LCM to procure specific cell populations. The manual exfoliation technique does not destroy the original tissue source, thereby allowing subsequent formalin tissue fixation. The technique of manual exfoliation in conjunction with LCM can enable the molecular profiling of a sampled selected cell population. Because it does not destroy the original tissue, histologic correlation can be combined with molecular profiling.
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Affiliation(s)
- Wilfrido D Mojica
- Department of Pathology, University at Buffalo, State University of New York, Buffalo, New York 14203, USA.
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Righetti PG, Castagna A, Antonucci F, Piubelli C, Cecconi D, Campostrini N, Rustichelli C, Antonioli P, Zanusso G, Monaco S, Lomas L, Boschetti E. Proteome analysis in the clinical chemistry laboratory: Myth or reality? Clin Chim Acta 2005; 357:123-39. [PMID: 15970281 DOI: 10.1016/j.cccn.2005.03.018] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2005] [Accepted: 03/09/2005] [Indexed: 11/26/2022]
Abstract
BACKGROUND We review here modern aspects of proteomic analysis, as displayed via orthogonal mass/charge analysis (isoelectric focusing in the first dimension, followed by sodium dodecyl sulphate electrophoresis in polyacrylamide gels, SDS-PAGE, at right angles, in the second dimension). METHODS This technique is capable of displaying a few thousand polypeptide chains, characterized by a single pI and M(r) value as coordinates, and recognized via elution, digestion and mass spectrometry analysis. Although, up to the present, this technique has been used mostly for advanced research, with no immediate applications in the clinical chemistry laboratory, there are hints that such applications will soon become a reality. RESULTS AND CONCLUSIONS In the field of cancer research, it is here shown that stathmin (Op18) becomes heavily phosphorylated in cancerous mantle cell lymphomas and that the progression of the disease can be followed by the progression of phosphorylation of Op18 and by the appearance of additional phosphorylated spots. Also chemoresistance of different tumors has been evaluated via 2D-PAGE through quantitative, differential proteomics: among up- and down-regulated proteins in a human cervix squamous cell carcinoma cell line (A431), rendered resistant to cisplatin, one particular protein was found to appear in large quantities by de novo synthesis: 14-3-3, a protein known to impart resistance to apoptosis to cells. In the field of brain disorders, we could set up an easy test for detecting pathological prions in sporadic Creutzfeldt-Jakob disease (sCJD), by simply searching for those pathological forms in the olfactory mucosa (up to this finding, diagnosis could only be confirmed post-mortem). We are currently working on a test for differentiating sCJD from all the other degenerative dementias. Upon 2D mapping of cerebrospinal fluid (CSF) and immunoblot analysis, we could identify a major spot (pI 4.8, M(r) 30 kDa) followed by some two-three minor spots (pIs 5.0-6.0, same M(r) value) of the same 14-3-3 anti-apoptotic protein involved in chemoresistance. By this test, sCJD could be differentiated from all the other degenerative dementias, which are 14-3-3 negative (in sCJD, the rapid and massive brain cell damage releases large quantities of 14-3-3 in the cerebrospinal fluid). Another protein that appears very promising as a marker for sCJD is cystatin C, that is strongly up-regulated in this pathology. Human sera should also be mined for discovery of many more markers for disease. Up to the present, no one could be found, but this was due to the presence of several major proteins, obscuring all rare ones. Via several immuno-subtraction steps, followed by ion exchange and size exclusion chromatography, one can now detect proteins and peptides present in sera at levels below 10 ng/mL, highlighting the road to discovery of novel markers of disease. Another technique that could revolutionize biomarker discovery in biological fluids consists in the use of combinatorial beads to reduce the dynamic range. They consist in a library of combinatorial ligands coupled to small beads. Such a library comprises hexameric ligands composed of amino acids, resulting in millions different structures. When these beads are impregnated with complex proteomes (e.g., human sera, CSF, urines) of widely differing protein compositions, they are able to significantly reduce the concentration differences, thus greatly enhancing the possibility of evidencing low-abundance species.
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Affiliation(s)
- Pier Giorgio Righetti
- Department of Agricultural and Industrial Biotechnologies, University of Verona, Strada Le Grazie No. 15, Verona 37134, Italy.
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Righetti PG, Castagna A, Antonucci F, Piubelli C, Cecconi D, Campostrini N, Antonioli P, Astner H, Hamdan M. Critical survey of quantitative proteomics in two-dimensional electrophoretic approaches. J Chromatogr A 2004; 1051:3-17. [PMID: 15532550 DOI: 10.1016/j.chroma.2004.05.106] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
The present review attempts to cover a number of methods that appeared in the last few years for performing quantitative proteome analysis. However, due to the large number of methods described for both electrophoretic and chromatographic approaches, we have limited this excursus only to conventional two-dimensional (2D) map analysis, coupling orthogonally a charge-based step (isoelectric focusing) to a size-based separation (sodium dodecyl sulfate (SDS)-electrophoresis). The first and oldest method applied in 2D mapping is based on statistical analysis performed on sets of gels via powerful software packages, such as the Melanie, PDQuest, Z3 and Z4000, Phoretix and Progenesis. This method calls for separately-running a number of replicas for control and treated samples, the merging and comparing between these two sets of data being accomplished via the softwares just mentioned. Recent developments permit analyses on a single gel containing mixed samples differentially labelled and resolved by either fluorescence or isotopic means. In one approach, a set of fluorophors, called Cy3 and Cy5, are selected for differentially tagging Lys residues, via a "minimal labelling" protocol. A variant of this, adopts a newer set of fluorophors, also of the Cy3 and Cy5 type, reacting on Cys residues, via a strategy of "saturation labelling". There are at present two methods for quantitative proteomics in a 2D gel format exploiting stable isotopes: one utilizes tagging Cys residues with [2H0]/[2H3]-acrylamide; the other one, also based on a Cys reactive compound, exploits [2H0]/[2H4] 2-vinylpyridine. The latter reagent achieves 100% efficiency coupled to 100% specificity. The advantages and limitations of the various protocols are discussed.
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Affiliation(s)
- Pier Giorgio Righetti
- Department of Agricultural and Industrial Biotechnologies, University of Verona, Verona, Italy.
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Fountoulakis M. Application of proteomics technologies in the investigation of the brain. MASS SPECTROMETRY REVIEWS 2004; 23:231-258. [PMID: 15133836 DOI: 10.1002/mas.10075] [Citation(s) in RCA: 85] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
Approximately 30-50% of the genes in mammals are expressed in the nervous system. A differential expression of genes in distinct patterns is necessary for the generation of the large variety of neuronal phenotypes. Proteomic analysis of brain compartments may be useful to understand the complexity, to investigate disorders of the central nervous system, and to search for corresponding early markers. Up to now, proteomics has mainly studied the identity and levels of the abundant human, rat, and mouse brain proteins as well as changes of their levels and the modifications that result from various neurological disorders, like Alzheimer's disease and Down's syndrome in humans and in animal models of those diseases. The proteins, for which altered levels in these disorders have been observed, exert mainly neurotransmission, guidance, and signal-transduction functions, or are involved in detoxification, metabolism, and conformational changes. Some of those proteins may be potential drug targets. Further improvement of proteomics technologies to increase sensitivity and efficiency of detection of certain protein classes is necessary for a more detailed analysis of the brain proteome. In this review, a description of the proteomics technologies applied in the investigation of the brain, the major findings that resulted from their application, and the potential and limitations of the current technologies are discussed.
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Affiliation(s)
- Michael Fountoulakis
- F. Hoffmann-La Roche Ltd., Center for Medical Genomics, Building 93-444, 4070 Basel, Switzerland.
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Chan-Hui PY, Stephens K, Warnock RA, Singh S. Applications of eTag™ assay platform to systems biology approaches in molecular oncology and toxicology studies. Clin Immunol 2004; 111:162-74. [PMID: 15137949 DOI: 10.1016/j.clim.2003.12.015] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2003] [Accepted: 12/23/2003] [Indexed: 02/02/2023]
Abstract
We have developed a universal eTag trade mark multiplex assay platform that can be uniquely applied to survey the molecule profiles of biologic systems in sub-global large-scale analyses. The effectiveness of eTag trade mark assays when applied to focused system biology studies in molecular oncology and predictive toxicology is herein described while reviewing the current methods commonly used. The multi-analyte and multi-parameter assay approach for parallel analysis will form the basis of an emerging paradigm of multiplexed molecular profiling for signaling pathway networks and various aspects of drug development processes.
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Affiliation(s)
- P-Y Chan-Hui
- Aclara BioSciences, Inc., Mountain View, CA 94043, USA
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37
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Bisca A, D'Ambrosio C, Scaloni A, Puglisi F, Aprile G, Piga A, Zuiani C, Bazzocchi M, Di Loreto C, Paron I, Tell G, Damante G. Proteomic evaluation of core biopsy specimens from breast lesions. Cancer Lett 2004; 204:79-86. [PMID: 14744537 DOI: 10.1016/j.canlet.2003.09.028] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Analysis of tumour samples by a proteomic technology, which combines two-dimensional gel electrophoresis and mass spectrometry analysis, is a promising approach for molecular characterization of cancer. Proteomic analysis of neoplasms is usually performed on surgical material. The possibility to perform proteomic analysis on pre-operative samples might be useful for diagnostic purposes or for determination of tumour sensitivity to therapy. In this study, we report how tissues from core biopsy of breast lesions can be routinely used to obtain accurate protein expression profiles by proteomic analysis. Protein profiles from fibroadenomas were compared to those from ductal infiltrating carcinomas. By using mass spectrometry, identification of proteins overexpressed in carcinomas with respect to fibroadenomas was obtained. Thus, our study provides a methodology to perform proteomic analysis on pre-operative samples of breast lesions.
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Affiliation(s)
- Alessia Bisca
- Dipartimento di Scienze e Tecnologie Biomediche, Centro M.A.T.I., Università di Udine, Piazzale Kolbe 1, Udine 33100, Italy
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Mocellin S, Rossi CR, Traldi P, Nitti D, Lise M. Molecular oncology in the post-genomic era: the challenge of proteomics. Trends Mol Med 2004; 10:24-32. [PMID: 14720583 DOI: 10.1016/j.molmed.2003.11.001] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Affiliation(s)
- Simone Mocellin
- Department of Oncological and Surgical Sciences, University of Padua, via Giustiniani 2, 35128 Padua, Italy.
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Abstract
Slow transforming retroviruses, such as the Moloney murine leukemia virus (M-MuLV), induce tumors upon infection of a host after a relatively long latency period. The underlying mechanism leading to cell transformation is the activation of proto-oncogenes or inactivation of tumor suppressor genes as a consequence of proviral insertions into the host genome. Cells carrying proviral insertions that confer a selective advantage will preferentially grow out. This means that proviral insertions mark genes contributing to tumorigenesis, as was demonstrated by the identification of numerous proto-oncogenes in retrovirally induced tumors in the past. Since cancer is a complex multistep process, the proviral insertions in one clone of tumor cells also represent oncogenic events that cooperate in tumorigenesis. Novel advances, such as the launch of the complete mouse genome, high-throughput isolation of proviral flanking sequences, and genetically modified animals have revolutionized proviral tagging into an elegant and efficient approach to identify signaling pathways that collaborate in cancer.
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Affiliation(s)
- Harald Mikkers
- Division of Molecular Genetics and Centre of Biomedical Genetics, Netherlands Cancer Institute 1066 CX, Amsterdam, The Netherlands
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Marengo E, Robotti E, Righetti PG, Antonucci F. New approach based on fuzzy logic and principal component analysis for the classification of two-dimensional maps in health and disease. Application to lymphomas. J Chromatogr A 2003; 1004:13-28. [PMID: 12929957 DOI: 10.1016/s0021-9673(03)00852-5] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Two-dimensional (2D) electrophoresis is the most wide spread technique for the separation of proteins in biological systems. This technique produces 2D maps of high complexity, which creates difficulties in the comparison of different samples. The method proposed in this paper for the comparison of different 2D maps can be summarised in four steps: (a) digitalisation of the image; (b) fuzzyfication of the digitalised map in order to consider the variability of the two-dimensional electrophoretic separation; (c) decoding by principal component analysis of the previously obtained fuzzy maps, in order to reduce the system dimensionality; (d) classification analysis (linear discriminant analysis), in order to separate the samples contained in the dataset according to the classes present in said dataset. This method was applied to a dataset constituted by eight samples: four belonging to healthy human lymph-nodes and four deriving from non-Hodgkin lymphomas. The amount of fuzzyfication of the original map is governed by the sigma parameter. The larger the value, the more fuzzy theresulting transformed map. The effect of the fuzzyfication parameter was investigated, the optimal results being obtained for sigma = 1.75 and 2.25. Principal component analysis and linear discriminant analysis allowed the separation of the two classes of samples without any misclassification.
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Affiliation(s)
- Emilio Marengo
- Dipartimento di Scienze e Tecnologie Avanzate, Università del Piemonte Orientale, 15100 Alessandria, Italy.
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Abbott RT, Tripp S, Perkins SL, Elenitoba-Johnson KSJ, Lim MS. Analysis of the PI-3-Kinase-PTEN-AKT pathway in human lymphoma and leukemia using a cell line microarray. Mod Pathol 2003; 16:607-12. [PMID: 12808067 DOI: 10.1097/01.mp.0000067423.83712.74] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Tissue microarray technology facilitates rapid assessment of expression of molecular markers by enabling the simultaneous analysis of hundreds of tissue specimens. We have applied this technology to establish a microarray composed of cell pellets derived from 40 human lymphoma/leukemia-derived cell lines harboring a variety of molecular abnormalities. The application of cell line microarrays to the assessment of biologic marker evaluation was validated by studying the immunohistochemical expression of PTEN and phosphorylated AKT, two mediators of the phosphatidylinositol (PI)-3-kinase pathway. In addition to the high throughout analysis of protein expression in lymphoma/leukemia cells, this methodology also enables the evaluation of subcellular localization of protein expression. Cytoplasmic PTEN expression was observed in the majority of cell lines (87%), whereas a minor subset demonstrated nuclear expression. Phosphorylated AKT was also expressed predominantly within the cytoplasm in 65% of cell lines, whereas nuclear expression was seen in a minority. An inverse relationship between PTEN and phosphorylated AKT was observed in 63% of cell lines. No cell lines showed absence of PTEN expression, whereas 50% of cell lines showed low PTEN expression. Our data support the integrity of the PI-3-kinase-PTEN-AKT pathway in a majority of cell lines derived from hematologic malignancies and clearly demonstrates the utility of microarray technology in the in situ assessment of expression of molecular markers in tumor-derived cell lines.
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Affiliation(s)
- Robert T Abbott
- Division of Anatomic Pathology, Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA
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Savchenko A, Yee A, Khachatryan A, Skarina T, Evdokimova E, Pavlova M, Semesi A, Northey J, Beasley S, Lan N, Das R, Gerstein M, Arrowmith CH, Edwards AM. Strategies for structural proteomics of prokaryotes: Quantifying the advantages of studying orthologous proteins and of using both NMR and X-ray crystallography approaches. Proteins 2003; 50:392-9. [PMID: 12557182 DOI: 10.1002/prot.10282] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Only about half of non-membrane-bound proteins encoded by either bacterial or archaeal genomes are soluble when expressed in Escherichia coli (Yee et al., Proc Natl Acad Sci USA 2002;99:1825-1830; Christendat et al., Prog Biophys Mol Biol 200;73:339-345). This property limits genome-scale functional and structural proteomics studies, which depend on having a recombinant, soluble version of each protein. An emerging strategy to increase the probability of deriving a soluble derivative of a protein is to study different sequence homologues of the same protein, including representatives from thermophilic organisms, based on the assumption that the stability of these proteins will facilitate structural analysis. To estimate the relative merits of this strategy, we compared the recombinant expression, solubility, and suitability for structural analysis by NMR and/or X-ray crystallography for 68 pairs of homologous proteins from E. coli and Thermotoga maritima. A sample suitable for structural studies was obtained for 62 of the 68 pairs of homologs under standardized growth and purification procedures. Fourteen (eight E. coli and six T. maritima proteins) samples generated NMR spectra of a quality suitable for structure determination and 30 (14 E. coli and 16 T. maritima proteins) samples formed crystals. Only three (one E. coli and two T. maritima proteins) samples both crystallized and had excellent NMR properties. The conclusions from this work are: (1) The inclusion of even a single ortholog of a target protein increases the number of samples for structural studies almost twofold; (2) there was no clear advantage to the use of thermophilic proteins to generate samples for structural studies; and (3) for the small proteins analyzed here, the use of both NMR and crystallography approaches almost doubled the number of samples for structural studies.
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Affiliation(s)
- Alexei Savchenko
- Ontario Center for Structural Proteomics, University Health Network, Toronto, Ontario, Canada
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Abstract
The advent of proteomics techniques has been enthusiastically accepted in most areas of biology and medicine. In neuroscience, a host of applications was proposed ranging from neurotoxicology, neurometabolism, determination of the proteome of the individual brain areas in health and disease, to name a few. Only recently, the limitations of the method have been shown, hampering the rapid spreading of the technology, which in principle consists of two-dimensional gel electrophoresis with in-gel protein digestion of protein spots and identification by mass-spectrometrical approaches or microsequencing. The identification, including quantification using specific software, of brain protein classes, like enzymes, cytoskeleton proteins, heat shock proteins/chaperones, proteins of the transcription and translation machinery, synaptosomal proteins, antioxidant proteins, is a clear domain of proteomics. Furthermore, the concomitant detection of several hundred proteins on a gel allows the demonstration of an expressional pattern, rather generated by a reliable, protein-chemical method than by immunoreactivity, proposed by protein-arrays. An additional advantage is that hitherto unknown proteins, so far only proposed from their nucleic acid structure, designated as hypothetical proteins, can be identified as brain proteins. As to shortcomings and disadvantages of the method we would point to the major problem, the failure to separate hydrophobic proteins. There is so far no way to analyse the vast majority of these proteins in gels. Several other analytical problems need to be overcome, but once the latter problem can be solved, there is nothing to stop the method for a large scale analysis of membrane proteins in neuroscience.
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Affiliation(s)
- Gert Lubec
- Department of Pediatrics, University of Vienna, Währinger Gürtel 18, A 1090, Vienna, Austria.
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Aksu S, Scheler C, Focks N, Leenders F, Theuring F, Salnikow J, Jungblut PR. An iterative calibration method with prediction of post-translational modifications for the construction of a two-dimensional electrophoresis database of mouse mammary gland proteins. Proteomics 2002; 2:1452-63. [PMID: 12422362 DOI: 10.1002/1615-9861(200210)2:10<1452::aid-prot1452>3.0.co;2-n] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Protein databases serve as general reference resources providing an orientation on two-dimensional electrophoresis (2-DE) patterns of interest. The intention behind constructing a 2-DE database of the water soluble proteins from wild-type mouse mammary gland tissue was to create a reference before going on to investigate cancer-associated protein variations. This database shall be deemed to be a model system for mouse tissue, which is open for transgenic or knockout experiments. Proteins were separated and characterized in terms of their molecular weight (M(r)) and isoelectric point (pI) by high resolution 2-DE. The proteins were identified using prevalent proteomics methods. One method was peptide mass fingerprinting by matrix-assisted laser desorption/ionization-mass spectrometry. Another method was N-terminal sequencing by Edman degradation. By N-terminal sequencing M(r) and pI values were specified more accurately and so the calibration of the master gel was obtained more systematically and exactly. This permits the prediction of possible post-translational modifications of some proteins. The mouse mammary gland 2-DE protein database created presently contains 66 identified protein spots, which are clickable on the gel pattern. This relational database is accessible on the WWW under the URL: http://www.mpiib-berlin.mpg.de/2D-PAGE.
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Affiliation(s)
- Sevil Aksu
- Technical University, Max-Volmer Institute for Biophysical Chemistry and Biochemistry, Berlin, Germany
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45
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Abstract
Drug discovery is a prolonged process that uses a variety of tools from diverse fields. To accelerate the process, a number of biotechnologies, including genomics, proteomics and a number of cellular and organismic methodologies, have been developed. Proteomics development faces interdisciplinary challenges, including both the traditional (biology and chemistry) and the emerging (high-throughput automation and bioinformatics). Emergent technologies include two-dimensional gel electrophoresis, mass spectrometry, protein arrays, isotope-encoding, two-hybrid systems, information technology and activity-based assays. These technologies, as part of the arsenal of proteomics techniques, are advancing the utility of proteomics in the drug-discovery process.
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Affiliation(s)
- Jonathan Burbaum
- ActivX Biosciences, Inc., 11025 North Torrey Pines Road, Suite 120, La Jolla, CA 92037, USA.
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Dangles V, Lazar V, Validire P, Richon S, Wertheimer M, Laville V, Janneau JL, Barrois M, Bovin C, Poynard T, Vallancien G, Bellet D. Gene expression profiles of bladder cancers: evidence for a striking effect of in vitro cell models on gene patterns. Br J Cancer 2002; 86:1283-9. [PMID: 11953886 PMCID: PMC2375349 DOI: 10.1038/sj.bjc.6600239] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2001] [Revised: 01/22/2002] [Accepted: 02/19/2002] [Indexed: 11/24/2022] Open
Abstract
In order to assess the effect of in vitro models on the expression of key genes known to be implicated in the development or progression of cancer, we quantified by real-time quantitative PCR the expression of 28 key genes in three bladder cancer tissue specimens and in their derived cell lines, studied either as one-dimensional single cell suspensions, two-dimensional monolayers or three-dimensional spheroids. Global analysis of gene expression profiles showed that in vitro models had a dramatic impact upon gene expression. Remarkably, quantitative differences in gene expression of 2-63-fold were observed in 24 out of 28 genes among the cell models. In addition, we observed that the in vitro model which most closely mimicked in vivo mRNA phenotype varied with both the gene and the patient. These results provide evidence that mRNA expression databases based on cancer cell lines, which are studied to provide a rationale for selection of therapy on the basis of molecular characteristics of a patient's tumour, must be carefully interpreted.
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Affiliation(s)
- V Dangles
- Laboratoire d'Immunologie des Tumeurs, ESA 8067 CNRS, Faculté des Sciences Pharmaceutiques et Biologiques de Paris, Université Paris V-René Descartes, 4 avenue de l'Observatoire, 75006 Paris, France
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Current Awareness on Comparative and Functional Genomics. Comp Funct Genomics 2002. [PMCID: PMC2447281 DOI: 10.1002/cfg.118] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
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Chang JW, Jeon HB, Lee JH, Yoo JS, Chun JS, Kim JH, Yoo YJ. Augmented expression of peroxiredoxin I in lung cancer. Biochem Biophys Res Commun 2001; 289:507-12. [PMID: 11716502 DOI: 10.1006/bbrc.2001.5989] [Citation(s) in RCA: 96] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Comparative proteome analysis was performed between human normal (BEAS 2B) and malignant (A549) lung epithelial cells in an attempt to identify novel biomarkers of lung cancer. Approximately 500 protein spots could be separated by mini two-dimensional electrophoresis and visualized with Coomassie blue R-250. Among those relatively abundant proteins, eight spots were changed more than twofold reproducibly and identified by peptide mass fingerprints using mass spectrometry and database search. The increased proteins in A549 were aldehyde dehydrogenase, peroxiredoxin I, fatty acid binding protein, aldoketoreductase, and destrin, whereas the decreased proteins were galectin-1, transgelin, and stathmin. Since human lung is exposed to continuous oxidative stress, antioxidant enzyme peroxiredoxin I was selected for further investigation and its augmented expression was confirmed in cancer tissues compared to normal tissues from lung cancer patients, suggesting peroxiredoxin I as a potential biomarker of lung cancer.
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Affiliation(s)
- J W Chang
- Department of Life Science, Kwangju Institute of Science and Technology (K-JIST), Kwangju 500-712, Korea
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50
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