1
|
Li Y, Wang S, Han C, Li XL, Min JZ. Unlocking the future of colorectal cancer detection: Advances in screening glycosylation-based biomarkers on biological mass spectrometry technology. J Chromatogr A 2024; 1738:465501. [PMID: 39504704 DOI: 10.1016/j.chroma.2024.465501] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2024] [Revised: 10/31/2024] [Accepted: 11/02/2024] [Indexed: 11/08/2024]
Abstract
The incidence of colorectal cancer (CRC) is increasingly affecting younger populations, with its mortality rate rising annually. However, current clinical diagnostic techniques, such as colonoscopy and CEA antigen testing, remain invasive and prone to false-positive results, complicating early diagnosis and intervention. Glycosylation, a key post-translational modification, plays an essential role in cellular function, physiological regulation, and disease processes. In recent years, mass spectrometry technology has emerged as a powerful tool for screening glycan biomarkers, owing to its exceptional separation capabilities and sensitivity. This review encompasses the advancements in CRC glycan biomarkers from 2016 to 2024, with particular emphasis placed on N/O-glycan biomarkers identified through mass spectrometry. Nonetheless, the intrinsic low abundance and polyhydroxy nature of glycans hinder the specificity and sensitivity of current glycan biomarkers. To overcome these limitations, this article outlines pretreatment strategies for N/O-glycans, including glycan release, enrichment, purification, and derivatization, in conjunction with relative quantification techniques and high-throughput bioinformatics tools for biomarker screening. These strategies are anticipated to enhance the efficiency and precision of glycan biomarker identification through mass spectrometry. These advancements hold significant promise for enhancing CRC prevention, diagnosis, and treatment, thereby potentially improving patient outcomes and quality of life.
Collapse
Affiliation(s)
- Yuxuan Li
- Key Laboratory of Natural Medicines of the Changbai Mountain, Ministry of Education, Department of Pharmaceutical Analysis, College of Pharmacy Yanbian University, Yanji, 133002, Jilin Province, China
| | - Songze Wang
- Key Laboratory of Natural Medicines of the Changbai Mountain, Ministry of Education, Department of Pharmaceutical Analysis, College of Pharmacy Yanbian University, Yanji, 133002, Jilin Province, China
| | - Chengqiang Han
- Key Laboratory of Natural Medicines of the Changbai Mountain, Ministry of Education, Department of Pharmaceutical Analysis, College of Pharmacy Yanbian University, Yanji, 133002, Jilin Province, China
| | - Xi-Ling Li
- Key Laboratory of Natural Medicines of the Changbai Mountain, Ministry of Education, Department of Pharmaceutical Analysis, College of Pharmacy Yanbian University, Yanji, 133002, Jilin Province, China
| | - Jun Zhe Min
- Key Laboratory of Natural Medicines of the Changbai Mountain, Ministry of Education, Department of Pharmaceutical Analysis, College of Pharmacy Yanbian University, Yanji, 133002, Jilin Province, China.
| |
Collapse
|
2
|
Liu C, Otsuka K, Kawai T. Recent advances in microscale separation techniques for glycome analysis. J Sep Sci 2024; 47:e2400170. [PMID: 38863084 DOI: 10.1002/jssc.202400170] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2024] [Revised: 05/12/2024] [Accepted: 05/24/2024] [Indexed: 06/13/2024]
Abstract
The glycomic analysis holds significant appeal due to the diverse roles that glycans and glycoconjugates play, acting as modulators and mediators in cellular interactions, cell/organism structure, drugs, energy sources, glyconanomaterials, and more. The glycomic analysis relies on liquid-phase separation technologies for molecular purification, separation, and identification. As a miniaturized form of liquid-phase separation technology, microscale separation technologies offer various advantages such as environmental friendliness, high resolution, sensitivity, fast speed, and integration capabilities. For glycan analysis, microscale separation technologies are continuously evolving to address the increasing challenges in their unique manners. This review discusses the fundamentals and applications of microscale separation technologies for glycomic analysis. It covers liquid-phase separation technologies operating at scales generally less than 100 µm, including capillary electrophoresis, nanoflow liquid chromatography, and microchip electrophoresis. We will provide a brief overview of glycomic analysis and describe new strategies in microscale separation and their applications in glycan analysis from 2014 to 2023.
Collapse
Affiliation(s)
- Chenchen Liu
- Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Japan
| | - Koji Otsuka
- Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan
- Research Administration Center, Osaka Metropolitan University, Osaka, Japan
| | - Takayuki Kawai
- Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Japan
- RIKEN Center for Biosystems Dynamics Research, Osaka, Japan
| |
Collapse
|
3
|
Pongracz T, Mayboroda OA, Wuhrer M. The Human Blood N-Glycome: Unraveling Disease Glycosylation Patterns. JACS AU 2024; 4:1696-1708. [PMID: 38818049 PMCID: PMC11134357 DOI: 10.1021/jacsau.4c00043] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/10/2024] [Revised: 03/28/2024] [Accepted: 04/01/2024] [Indexed: 06/01/2024]
Abstract
Most of the proteins in the circulation are N-glycosylated, shaping together the total blood N-glycome (TBNG). Glycosylation is known to affect protein function, stability, and clearance. The TBNG is influenced by genetic, environmental, and metabolic factors, in part epigenetically imprinted, and responds to a variety of bioactive signals including cytokines and hormones. Accordingly, physiological and pathological events are reflected in distinct TBNG signatures. Here, we assess the specificity of the emerging disease-associated TBNG signatures with respect to a number of key glycosylation motifs including antennarity, linkage-specific sialylation, fucosylation, as well as expression of complex, hybrid-type and oligomannosidic N-glycans, and show perplexing complexity of the glycomic dimension of the studied diseases. Perspectives are given regarding the protein- and site-specific analysis of N-glycosylation, and the dissection of underlying regulatory layers and functional roles of blood protein N-glycosylation.
Collapse
Affiliation(s)
- Tamas Pongracz
- Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333ZA Leiden, The Netherlands
| | - Oleg A. Mayboroda
- Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333ZA Leiden, The Netherlands
| | - Manfred Wuhrer
- Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333ZA Leiden, The Netherlands
| |
Collapse
|
4
|
Young TW, Kappler MP, Hockaden NM, Carpenter RL, Jacobson SC. Characterization of Extracellular Vesicles by Resistive-Pulse Sensing on In-Plane Multipore Nanofluidic Devices. Anal Chem 2023; 95:16710-16716. [PMID: 37916500 PMCID: PMC10841850 DOI: 10.1021/acs.analchem.3c03546] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2023]
Abstract
Extracellular vesicles (EVs) are cell-derived, naturally produced, membrane-bound nanoscale particles that are linked to cell-cell communication and the propagation of diseases. Here, we report the design and testing of in-plane nanofluidic devices for resistive-pulse measurements of EVs derived from bovine milk and human breast cancer cells. The devices were fabricated in plane with three nanopores in series to determine the particle volume and diameter, two pore-to-pore regions to measure the electrophoretic mobility and zeta potential, and an in-line filter to prevent cellular debris and aggregates from entering the nanopore region. Devices were tested with and without the channels coated with a short-chain PEG silane to minimize electroosmotic flow and permit an accurate measurement of the electrophoretic mobility and zeta potential of the EVs. To enhance throughput of EVs, vacuum was applied to the waste reservoir to increase particle frequencies up to 1000 min-1. The nanopores had cross-sections 200 nm wide and 200 nm deep and easily resolved EV diameters from 60 to 160 nm. EVs from bovine milk and human breast cancer cells had similar particle size distributions, but their zeta potentials differed by 2-fold, -8 ± 1 and -4 ± 1 mV, respectively.
Collapse
Affiliation(s)
- Tanner W Young
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, United States
| | - Michael P Kappler
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, United States
| | - Natasha M Hockaden
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine-Bloomington, Bloomington, Indiana 47405-7005, United States
| | - Richard L Carpenter
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine-Bloomington, Bloomington, Indiana 47405-7005, United States
| | - Stephen C Jacobson
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102, United States
| |
Collapse
|
5
|
Nimbkar S, Leena MM, Moses JA, Anandharamakrishnan C. Microfluidic assessment of nutritional biomarkers: Concepts, approaches and advances. Crit Rev Food Sci Nutr 2022; 64:5113-5127. [PMID: 36503314 DOI: 10.1080/10408398.2022.2150597] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Among various approaches to understand the health status of an individual, nutritional biomarkers can provide valuable information, particularly in terms of deficiencies, if any, and their severity. Commonly, the approach revolves around molecular sciences, and the information gained can support prognosis, diagnosis, remediation, and impact assessment of therapies. Microfluidic platforms can offer benefits of low sample and reagent requirements, low cost, high precision, and lower detection limits, with simplicity in handling and the provision for complete automation and integration with information and communication technologies (ICTs). While several advances are being made, this work details the underlying concepts, with emphasis on different point-of-care devices for the analysis of macro and micronutrient biomarkers. In addition, the scope of using different wearable microfluidic sensors for real-time and noninvasive determination of biomarkers is detailed. While several challenges remain, a strong focus is given on recent advances, presenting the state-of-the-art of this field. With more such biomarkers being discovered and commercialization-driven research, trends indicate the wide prospects of this advancing field in supporting clinicians, food technologists, nutritionists, and others.
Collapse
Affiliation(s)
- Shubham Nimbkar
- Computational Modeling and Nanoscale Processing Unit, National Institute of Food Technology, Entrepreneurship and Management, Ministry of Food Processing Industries, Thanjavur, Tamil Nadu, India
| | - M Maria Leena
- Computational Modeling and Nanoscale Processing Unit, National Institute of Food Technology, Entrepreneurship and Management, Ministry of Food Processing Industries, Thanjavur, Tamil Nadu, India
| | - Jeyan Arthur Moses
- Computational Modeling and Nanoscale Processing Unit, National Institute of Food Technology, Entrepreneurship and Management, Ministry of Food Processing Industries, Thanjavur, Tamil Nadu, India
| | - Chinnaswamy Anandharamakrishnan
- Computational Modeling and Nanoscale Processing Unit, National Institute of Food Technology, Entrepreneurship and Management, Ministry of Food Processing Industries, Thanjavur, Tamil Nadu, India
| |
Collapse
|
6
|
Zhou X, Song W, Novotny MV, Jacobson SC. Fractionation and characterization of sialyl linkage isomers of serum N-glycans by CE-MS. J Sep Sci 2022; 45:3348-3361. [PMID: 35819141 PMCID: PMC9473921 DOI: 10.1002/jssc.202200223] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2022] [Revised: 06/12/2022] [Accepted: 07/07/2022] [Indexed: 11/07/2022]
Abstract
Structural isomers of sialylated N-glycans contribute to the diversity of the N-glycome and to a range of biological functions. Sialyl linkage isomers can be readily distinguished by mass spectrometry with mass differences between α2,3- and α2,6-linkages generated by a two-step sialic acid linkage-specific alkylamidation. To improve the identification of N-glycans from complex mixtures, we added a delactonization step after the first alkylamidation step, which regenerates negatively charged carboxylic acids on α2,3-sialic acids. N-glycan isomers with α2,3-sialic acids are then fractionated by ion-exchange chromatography prior to the second alkylamidation step. With this modified alkylamidation method, sialylated N-glycans were enriched and stabilized for structural characterization by capillary electrophoresis-mass spectrometry and tandem mass spectrometry. We identified 52 sialylated N-glycan structures, including 107 linkage isomers, in human serum and confirmed the presence of positional isomers of specific sialyl linkage isomers. Due to the reduced sample complexity after ion-exchange fractionation and CE separation, substructural features of N-glycans were rapidly evaluated and included core- and antenna-fucosylation and poly-lactosamine.
Collapse
Affiliation(s)
- Xiaomei Zhou
- Department of Chemistry, Indiana University, Bloomington, Indiana 47401-7102, U.S.A
| | - Woran Song
- Department of Chemistry, Indiana University, Bloomington, Indiana 47401-7102, U.S.A
| | - Milos V. Novotny
- Department of Chemistry, Indiana University, Bloomington, Indiana 47401-7102, U.S.A
| | - Stephen C. Jacobson
- Department of Chemistry, Indiana University, Bloomington, Indiana 47401-7102, U.S.A
| |
Collapse
|
7
|
Wang HJ, Xie YB, Zhang PJ, Jiang T. Evaluation of the diagnostic value of serum-based proteomics for colorectal cancer. World J Gastrointest Oncol 2022; 14:1562-1573. [PMID: 36160749 PMCID: PMC9412932 DOI: 10.4251/wjgo.v14.i8.1562] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Revised: 04/13/2022] [Accepted: 07/18/2022] [Indexed: 02/05/2023] Open
Abstract
BACKGROUND Colorectal cancer (CRC) is a highly malignant cancer with a high incidence and mortality in China. It is urgent to find a diagnostic marker with higher sensitivity and specificity than the traditional approaches for CRC diagnosis.
AIM To provide new ideas for the diagnosis of CRC based on serum proteomics.
METHODS Specimens from 83 healthy people, 62 colon polyp (CRP) patients, and 101 CRC patients were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The diagnostic value of the profiles of differentially expressed proteins was then analyzed.
RESULTS Compared with the healthy control group, CRC patients had elevated expression of 5 proteins and reduced expression of 14 proteins. The area under the curve (AUC) for a differentially expressed protein with a mass-to-charge ratio of 2022.34 was the largest; the AUC was 0.843, which was higher than the AUC of 0.717 observed with carcinoembryonic antigen (CEA), and the sensitivity and specificity of this identified marker were 75.3% and 79.5%, respectively. After cross-validation, the accuracy of diagnosis using levels of this differentially expressed protein was 82.37%. Compared with the CRP group, the expression of 3 proteins in the serum of CRC patients was elevated and 11 proteins were expressed at reduced levels. Proteins possessing mass-to-charge ratio values of 2899.38 and 877.3 were selected to establish a classification tree model. The results showed that the accuracy of CRC diagnosis was 89.5%, the accuracy of CRP diagnosis was 81.6%, and the overall accuracy of this approach was 86.3%. The overall sensitivity and specificity of diagnosis using the proteomics approach were 81.8% and 66.75%, respectively. The sensitivities and specificities of diagnoses based on CEA and carbohydrate antigen 19-9 expression were 55.6% and 91.3% and 65.4% and 65.2%, respectively.
CONCLUSION We demonstrated that serum proteomics may be helpful for the detection of CRC, and it may assist clinical practice for CRC diagnosis.
Collapse
Affiliation(s)
- Hui-Juan Wang
- Department of Respiratory and Critical Care Medicine, Beijing Chao-Yang Hospital, Beijing Institute of Respiratory Medicine, Capital Medical University, Beijing 100020, China
| | - Yi-Bin Xie
- Department of Pancreatic and Gastric Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China
| | - Peng-Jun Zhang
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Interventional Therapy Department, Peking University Cancer Hospital and Institute, Beijing 100142, China
| | - Tao Jiang
- Medical Innovation Research Division of Chinese PLA General Hospital, Beijing 100853, China
| |
Collapse
|
8
|
Lin Q, Fang X, Chen H, Weng W, Liu B, Kong J. Dual-modality loop-mediated isothermal amplification for pretreatment-free detection of Septin9 methylated DNA in colorectal cancer. Mikrochim Acta 2021; 188:307. [PMID: 34453211 PMCID: PMC8396143 DOI: 10.1007/s00604-021-04979-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2021] [Accepted: 08/07/2021] [Indexed: 12/25/2022]
Abstract
Currently, the determination of DNA methylation is still a challenge due to the limited efficiency of enrichment, bisulfite modification, and detection. In this study, a dual-modality loop-mediated isothermal amplification integrated with magnetic bead isolation is proposed for the determination of methylated Septin9 gene in colorectal cancer. Magnetic beads modified with anti-methyl cytosine antibody were prepared for fast enrichment of methylated DNA through specific immunoaffinity (30 min). One-pot real-time fluorescence and colorimetric loop-mediated isothermal amplification were simultaneously developed for detecting methylated Septin9 gene (60 min). The real-time fluorescence generating by SYTO-9 dye (excitation: 470 nm and emission: 525 nm) and pH indicator (neutral red) was used for quantitative and visualized detection of methylated DNA. This method was demonstrated to detect methylated DNA from HCT 116 cells ranging from 2 to 0.02 ng/μL with a limit of detection of 0.02 ± 0.002 ng/μL (RSD: 9.75%). This method also could discriminate methylated Septin9 in 0.1% HCT 116 cells (RSD: 6.60%), suggesting its high specificity and sensitivity. The feasibility of this assay was further evaluated by clinical plasma samples from 20 colorectal cancer patients and 20 healthy controls, which shows the potential application in simple, low cost, quantitative, and visualized detection of methylated nucleic acids.
Collapse
Affiliation(s)
- Qiuyuan Lin
- Department of Chemistry, Fudan University, Shanghai, 200438, People's Republic of China
| | - Xueen Fang
- Department of Chemistry, Fudan University, Shanghai, 200438, People's Republic of China
| | - Hui Chen
- Department of Chemistry, Fudan University, Shanghai, 200438, People's Republic of China.
| | - Wenhao Weng
- Department of Clinical Laboratory, Yangpu Hospital, Tongji University School of Medicine, Shanghai, 200090, China.
| | - Baohong Liu
- Department of Chemistry, Fudan University, Shanghai, 200438, People's Republic of China
- Shanghai Stomatological Hospital, Shanghai, 200438, People's Republic of China
| | - Jilie Kong
- Department of Chemistry, Fudan University, Shanghai, 200438, People's Republic of China.
| |
Collapse
|
9
|
Harvey DJ. ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION MASS SPECTROMETRY: AN UPDATE FOR 2015-2016. MASS SPECTROMETRY REVIEWS 2021; 40:408-565. [PMID: 33725404 DOI: 10.1002/mas.21651] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/06/2020] [Accepted: 07/24/2020] [Indexed: 06/12/2023]
Abstract
This review is the ninth update of the original article published in 1999 on the application of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2016. Also included are papers that describe methods appropriate to analysis by MALDI, such as sample preparation techniques, even though the ionization method is not MALDI. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation and arrays. The second part of the review is devoted to applications to various structural types such as oligo- and poly-saccharides, glycoproteins, glycolipids, glycosides and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions and applications to chemical synthesis. The reported work shows increasing use of combined new techniques such as ion mobility and the enormous impact that MALDI imaging is having. MALDI, although invented over 30 years ago is still an ideal technique for carbohydrate analysis and advancements in the technique and range of applications show no sign of deminishing. © 2020 Wiley Periodicals, Inc.
Collapse
Affiliation(s)
- David J Harvey
- Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom
| |
Collapse
|
10
|
Coura MDM, Barbosa EA, Brand GD, Bloch C, de Sousa JB. Identification of Differential N-Glycan Compositions in the Serum and Tissue of Colon Cancer Patients by Mass Spectrometry. BIOLOGY 2021; 10:biology10040343. [PMID: 33923867 PMCID: PMC8074232 DOI: 10.3390/biology10040343] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/18/2021] [Revised: 03/29/2021] [Accepted: 04/06/2021] [Indexed: 01/08/2023]
Abstract
Simple Summary Incidence of colorectal cancer (CRC) has been rising in Brazil. To date, no reliable biomarker has been described in CRC for diagnosis and prognosis. Modifications in the N-glycosylation profile are usually associated with many cancers, as CRC. In turn, mass spectrometry (MS)-based methods are the most accurate technology in quantification of N-glycans. Therefore, we described a unique pattern of compositions altered in serum and tissues of stages II and III colon cancer patients, identified by MALDI-TOF/MS and LC-MS technology. N-glycans were mostly found decreased in serum whilst oligomannosidic, hypogalactosylated, and tetra-antennary forms were overexpressed in tumor tissues. Total N-glycome in serum of cancer patients was different from the profile found in serum of healthy individuals. Strikingly, no correlation between tissue N-glycosylation profile and serum profile was observed in cancer patients, posing the question where these compositions are originated from. Abstract Colorectal cancer (CRC) ranks second as the leading cause of cancer-related deaths worldwide. N-glycosylation is one of the most common posttranslational protein modifications. Therefore, we studied the total serum N-glycome (TSNG) of 13 colon cancer patients compared to healthy controls using MALDI-TOF/MS and LC-MS. N-glycosylation of cancer tumor samples from the same cohort were further quantified using a similar methodology. In total, 23 N-glycan compositions were down-regulated in the serum of colon cancer patients, mostly galactosylated forms whilst the mannose-rich HexNAc2Hex7, the fucosylated bi-antennary glycan HexNAc4Hex5Fuc1NeuAc2, and the tetra-antennary HexNAc6Hex7NeuAc3 were up-regulated in serum. Hierarchical clustering analysis of TSNG correctly singled out 85% of the patients from controls. Albeit heterogenous, N-glycosylation of tumor samples showed overrepresented oligomannosidic, bi-antennary hypogalactosylated, and branched compositions related to normal colonic tissue, in both MALDI-TOF/MS and LC-MS analysis. Moreover, compositions found upregulated in tumor tissue were mostly uncorrelated to compositions in serum of cancer patients. Mass spectrometry-based N-glycan profiling in serum shows potential in the discrimination of patients from healthy controls. However, the compositions profile in serum showed no parallel with N-glycans in tumor microenvironment, which suggests a different origin of compositions found in serum of cancer patients.
Collapse
Affiliation(s)
- Marcelo de M.A. Coura
- Division of Colorectal Surgery, University Hospital of Brasilia, School of Medicine, University of Brasilia, SGAN 605, Brasilia-DF 70840-901, Brazil;
- Correspondence:
| | - Eder A. Barbosa
- Laboratory of Mass Spectrometry, EMBRAPA Genetic Resources and Biotechnology, Parque Estação Biológica, PqEB, Av. W5 Norte, Brasilia-DF 70770-917, Brazil; (E.A.B.); (C.B.J.)
- Laboratory for the Synthesis and Analysis of Biomolecules, Institute of Chemistry, Campus Universitario Darcy Ribeiro, University of Brasilia, Brasilia-DF 70910-900, Brazil;
| | - Guilherme D. Brand
- Laboratory for the Synthesis and Analysis of Biomolecules, Institute of Chemistry, Campus Universitario Darcy Ribeiro, University of Brasilia, Brasilia-DF 70910-900, Brazil;
| | - Carlos Bloch
- Laboratory of Mass Spectrometry, EMBRAPA Genetic Resources and Biotechnology, Parque Estação Biológica, PqEB, Av. W5 Norte, Brasilia-DF 70770-917, Brazil; (E.A.B.); (C.B.J.)
| | - Joao B. de Sousa
- Division of Colorectal Surgery, University Hospital of Brasilia, School of Medicine, University of Brasilia, SGAN 605, Brasilia-DF 70840-901, Brazil;
| |
Collapse
|
11
|
Cajic S, Hennig R, Burock R, Rapp E. Capillary (Gel) Electrophoresis-Based Methods for Immunoglobulin (G) Glycosylation Analysis. EXPERIENTIA SUPPLEMENTUM (2012) 2021; 112:137-172. [PMID: 34687009 DOI: 10.1007/978-3-030-76912-3_4] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
The in-depth characterization of protein glycosylation has become indispensable in many research fields and in the biopharmaceutical industry. Especially knowledge about modulations in immunoglobulin G (IgG) N-glycosylation and their effect on immunity enabled a better understanding of human diseases and the development of new, more effective drugs for their treatment. This chapter provides a deeper insight into capillary (gel) electrophoresis-based (C(G)E) glycan analysis, addressing its impressive performance and possibilities, its great potential regarding real high-throughput for large cohort studies, as well as its challenges and limitations. We focus on the latest developments with respect to miniaturization and mass spectrometry coupling, as well as data analysis and interpretation. The use of exoglycosidase sequencing in combination with current C(G)E technology is discussed, highlighting possible difficulties and pitfalls. The application section describes the detailed characterization of N-glycosylation, utilizing multiplexed CGE with laser-induced fluorescence detection (xCGE-LIF). Besides a comprehensive overview on antibody glycosylation by comparing species-specific IgGs and human immunoglobulins A, D, E, G, and M, the chapter comprises a comparison of therapeutic monoclonal antibodies from different production cell lines, as well as a detailed characterization of Fab and Fc glycosylation. These examples illustrate the full potential of C(G)E, resolving the smallest differences in sugar composition and structure.
Collapse
Affiliation(s)
- Samanta Cajic
- Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany
| | - René Hennig
- Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany.
- glyXera GmbH, Magdeburg, Germany.
| | | | - Erdmann Rapp
- Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany
- glyXera GmbH, Magdeburg, Germany
| |
Collapse
|
12
|
Black AP, Angel PM, Drake RR, Mehta AS. Antibody Panel Based N-Glycan Imaging for N-Glycoprotein Biomarker Discovery. ACTA ACUST UNITED AC 2020; 98:e99. [PMID: 31721442 DOI: 10.1002/cpps.99] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Antibody panel based N-glycan imaging is a novel platform for N-glycan analysis of immunocaptured proteins. N-glycosylation is a post-translational modification of pathophysiological importance and is often studied in the context of disease biomarkers. Determination of protein-specific N-glycosylation changes in patient samples has traditionally been laborious or limited to study of a single protein per analysis. This novel technique allows for the multiplexed analysis of N-glycoproteins from biofluids. Briefly, this platform consists of antibodies spotted in an array panel to a microscope slide, specific capture of glycoproteins from a biological sample, and then enzymatic release of N-glycans for analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). N-glycans are detected at each individual spot, allowing N-glycan information to easily be linked back to its protein carrier. Using this protocol, multiplexed analysis of N-glycosylation on serum glycoproteins can be performed. Human serum is discussed here, but this method has potential to be applied to other biofluids and to any glycoprotein that can be captured by a validated antibody. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Antibody panel based N-glycan imaging by MALDI MS Support Protocol: Confirmation of antibody capture by IR-labeled proteins.
Collapse
Affiliation(s)
- Alyson P Black
- Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina
| | - Peggi M Angel
- Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina
| | - Richard R Drake
- Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina
| | - Anand S Mehta
- Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina
| |
Collapse
|
13
|
He C, Chen S, Zhao J, Tian J, Zhao S. Ultrasensitive detection of microRNA-21 based on electrophoresis assisted cascade chemiluminescence signal amplification for the identification of cancer cells. Talanta 2020; 209:120505. [DOI: 10.1016/j.talanta.2019.120505] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2019] [Revised: 10/21/2019] [Accepted: 10/24/2019] [Indexed: 02/08/2023]
|
14
|
Ou X, Chen P, Huang X, Li S, Liu B. Microfluidic chip electrophoresis for biochemical analysis. J Sep Sci 2019; 43:258-270. [DOI: 10.1002/jssc.201900758] [Citation(s) in RCA: 43] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2019] [Revised: 10/18/2019] [Accepted: 10/21/2019] [Indexed: 01/11/2023]
Affiliation(s)
- Xiaowen Ou
- Hubei Key Laboratory of Purification and Application of Plant Anti‐Cancer Active IngredientsCollege of Chemistry and Life ScienceHubei University of Education Wuhan P. R. China
- The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics‐Hubei Bioinformatics & Molecular Imaging Key LaboratorySystems Biology ThemeDepartment of Biomedical EngineeringCollege of Life Science and TechnologyHuazhong University of Science and Technology Wuhan P. R. China
| | - Peng Chen
- The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics‐Hubei Bioinformatics & Molecular Imaging Key LaboratorySystems Biology ThemeDepartment of Biomedical EngineeringCollege of Life Science and TechnologyHuazhong University of Science and Technology Wuhan P. R. China
| | - Xizhi Huang
- The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics‐Hubei Bioinformatics & Molecular Imaging Key LaboratorySystems Biology ThemeDepartment of Biomedical EngineeringCollege of Life Science and TechnologyHuazhong University of Science and Technology Wuhan P. R. China
| | - Shunji Li
- The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics‐Hubei Bioinformatics & Molecular Imaging Key LaboratorySystems Biology ThemeDepartment of Biomedical EngineeringCollege of Life Science and TechnologyHuazhong University of Science and Technology Wuhan P. R. China
| | - Bi‐Feng Liu
- The Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics‐Hubei Bioinformatics & Molecular Imaging Key LaboratorySystems Biology ThemeDepartment of Biomedical EngineeringCollege of Life Science and TechnologyHuazhong University of Science and Technology Wuhan P. R. China
| |
Collapse
|
15
|
Yang X, Zhao J, Chen S, Huang Y, Zhaok S. An ultrasensitive microchip electrophoresis chemiluminescence assay platform for detection of trace biomolecules. J Chromatogr A 2019; 1613:460693. [PMID: 31732154 DOI: 10.1016/j.chroma.2019.460693] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2019] [Revised: 11/05/2019] [Accepted: 11/07/2019] [Indexed: 11/19/2022]
Abstract
An ultrasensitive microchip electrophoresis chemiluminescence (MCE-CL) assay platform based on separation assisted cascade signal amplification was developed for detection of trace biomolecules. In this work, the aptamer was used as a target probe to bind target molecule and triggering cascade signal amplification reaction. The horseradish peroxide labeled DNA (HRP-DNA) was used as signal probe, utilizing nucleic acid hybridization and exonuclease cutting technology realized ultrasensitive detection of biomolecules on the MCE-CL assay platform. Taking gamma interferon (IFN-γ) as a model analyte, the linear range for IFN-γ detection is 8.0 × 10-15-1.0 × 10-8 M, the detection limit is 1.6 fM, which is six orders magnitude lower than that of without signal amplification. The proposed method was successfully applied for the quantification of IFN-γ in human plasma samples. It was demonstrated that the MCE-CL assay platform was quick, sensitive, and highly selective. It may serve as a tool for clinical analysis of IFN-γ to assist in the diagnosis of disease.
Collapse
Affiliation(s)
- Xing Yang
- State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Guangxi Normal University, Guilin 541004, China
| | - Jingjin Zhao
- State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Guangxi Normal University, Guilin 541004, China.
| | - Shengyu Chen
- State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Guangxi Normal University, Guilin 541004, China
| | - Yong Huang
- State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Guangxi Normal University, Guilin 541004, China
| | - Shulin Zhaok
- State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Guangxi Normal University, Guilin 541004, China.
| |
Collapse
|
16
|
Song W, Zhou X, Benktander JD, Gaunitz S, Zou G, Wang Z, Novotny MV, Jacobson SC. In-Depth Compositional and Structural Characterization of N-Glycans Derived from Human Urinary Exosomes. Anal Chem 2019; 91:13528-13537. [PMID: 31539226 PMCID: PMC6834888 DOI: 10.1021/acs.analchem.9b02620] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The study of exosomes has become increasingly popular due to their potentially important biological roles. Urine can be used as an effective source of exosomes for noninvasive investigations into the pathophysiological states of the urinary system, but first, detailed characterization of exosomal components in healthy individuals is essential. Here, we significantly extend the number of N-glycan compositions, including sulfated species, identified from urinary exosomes and determine the sialic acid linkages for many of those compositions. Capillary electrophoresis-mass spectrometry (CE-MS), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to identify N-glycan and sulfated N-glycan compositions. Second, because the alteration of sialylation patterns has been previously implicated in various disease states, ion-exchange chromatography, microfluidic capillary electrophoresis (CE), and MALDI-MS were adopted to resolve positional isomers of sialic acids. Structures of the sialyl-linkage isomers were assigned indirectly through α2-3 sialidase treatment and sialic acid linkage-specific alkylamidation (SALSA). In total, we have identified 219 N-glycan structures that include 175 compositions, 64 sialic acid linkage isomers, 26 structural isomers, and 27 sulfated glycans.
Collapse
Affiliation(s)
- Woran Song
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102
| | - Xiaomei Zhou
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102
| | - John D. Benktander
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102
| | - Stefan Gaunitz
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102
| | - Guozhang Zou
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102
| | - Ziyu Wang
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102
| | - Milos V. Novotny
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102
| | - Stephen C. Jacobson
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405-7102
| |
Collapse
|
17
|
Gebrehiwot AG, Melka DS, Kassaye YM, Gemechu T, Lako W, Hinou H, Nishimura SI. Exploring serum and immunoglobulin G N-glycome as diagnostic biomarkers for early detection of breast cancer in Ethiopian women. BMC Cancer 2019; 19:588. [PMID: 31208374 PMCID: PMC6580580 DOI: 10.1186/s12885-019-5817-8] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2018] [Accepted: 06/11/2019] [Indexed: 12/20/2022] Open
Abstract
BACKGROUND Alterations in protein glycosylation patterns have potentially been targeted for biomarker discovery in a wide range of diseases including cancer. Although there have been improvements in patient diagnosis and survival for breast cancer (BC), there is no clinically validated serum biomarker for its early diagnosis. Here, we profiled whole serum and purified Immunoglobulin G (IgG) fraction N-glycome towards identification of non-invasive glycan markers of BC. METHODS We employed a comprehensive glycomics approach by integrating glycoblotting-based glycan purification with MALDI-TOF/MS based quantitative analysis. Sera of BC patients belonging to stages I-IV and normal controls (NC) were collected from Ethiopian women during 2015-2016. IgG was purified by affinity chromatography using protein G spin plate and further subjected to glycoblotting for glycan release. Mass spectral data were further processed and evaluated rigorously, using various bioinformatics and statistical tools. RESULTS Out of 35 N-glycans that were significantly up-regulated in the sera of all BC patients compared to the NC, 17 complex type N-glycans showed profound expression abundance and diagnostic potential (AUC = 0.8-1) for the early stage (I and II) BC patients. Most of these glycans were core-fucosylated, multiply branched and sialylated structures, whose abundance has been strongly associated with greater invasive and metastatic potential of cancer. N-glycans quantified form IgG confirmed their abundance in BC patients, of which two core-fucosylated and agalactosylated glycans (m/z 1591, 1794) could specifically distinguish (AUC = 0.944 and 0.921, p ≤ 0.001) stage II patients from NC. Abundance of such structural features in IgG is associated with a decrease in its immunosuppressive potential towards tumor cells, which in part may correlate with the aggressive nature of BC commonly noticed in black population. CONCLUSIONS Our comprehensive study has addressed for the first time both whole serum and IgG N-glycosylation signatures of native black women suffering from BC and revealed novel glyco-biomarkers with marked overexpression and distinguishing ability at early stage patients. Further studies on direct identification of the intact glycoproteins using a glycoprteomics approach will provide a deeper understanding of specific biomarkers towards their clinical utility.
Collapse
Affiliation(s)
- Abrha G. Gebrehiwot
- Faculty of Advanced Life Science and Graduate School of Life Science, Hokkaido University, N21, W11, Kita-ku, Sapporo, 001-0021 Japan
| | - Daniel Seifu Melka
- Department of Biochemistry, School of Medicine, Addis Ababa University, Addis Ababa, Ethiopia
| | - Yimenashu Mamo Kassaye
- Department of Biochemistry, School of Medicine, Addis Ababa University, Addis Ababa, Ethiopia
| | - Tufa Gemechu
- Department of Pathology, School of Medicine, Addis Ababa University, Addis Ababa, Ethiopia
| | - Wajana Lako
- Department of Pathology, School of Medicine, Addis Ababa University, Addis Ababa, Ethiopia
| | - Hiroshi Hinou
- Faculty of Advanced Life Science and Graduate School of Life Science, Hokkaido University, N21, W11, Kita-ku, Sapporo, 001-0021 Japan
| | - Shin-Ichiro Nishimura
- Faculty of Advanced Life Science and Graduate School of Life Science, Hokkaido University, N21, W11, Kita-ku, Sapporo, 001-0021 Japan
| |
Collapse
|
18
|
Gizaw ST, Gaunitz S, Novotny MV. Highly Sensitive O-Glycan Profiling for Human Serum Proteins Reveals Gender-Dependent Changes in Colorectal Cancer Patients. Anal Chem 2019; 91:6180-6189. [PMID: 30983323 PMCID: PMC6602050 DOI: 10.1021/acs.analchem.9b00822] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
A newly developed microscale protocol for profiling serum O-glycans has been validated here with multiple serum samples obtained from different cohorts of colorectal cancer patients. The simultaneous cleavage and permethylation steps in this procedure preserve the integrity of released minor O-glycans, so that 39 O-linked oligosaccharides could be reliably recorded in a profile. This is far more detected components than shown in any previous studies. The analytical results were further subjected to a battery of statistical tests. Our O-glycan compositions compare favorably with the previous results obtained with solid tumors and cancer cell lines, suggesting that smaller circulatory mucins protruding into the blood circulation may be one source of O-glycans that we observe in the serum samples. While the control vs cancer statistical comparisons generally agree with the expected glycosylation trends, the comparisons of male vs female subjects have led to some surprising results for which we do not have a ready explanation due to lack of any literature describing hormonal control of O-glycosylation. Our results thus underscore the necessity of applying new analytical technologies to clinically interesting sample sets.
Collapse
Affiliation(s)
- Solomon T. Gizaw
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405
| | - Stefan Gaunitz
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405
| | - Milos V. Novotny
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405
| |
Collapse
|
19
|
Zhang Y, Zhao J, Chen S, Li S, Zhao S. A novel microchip electrophoresis laser induced fluorescence detection method for the assay of T4 polynucleotide kinase activity and inhibitors. Talanta 2019; 202:317-322. [PMID: 31171188 DOI: 10.1016/j.talanta.2019.05.032] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2019] [Revised: 05/01/2019] [Accepted: 05/04/2019] [Indexed: 12/19/2022]
Abstract
T4 polynucleotide kinase (T4 PNK) may catalyze the phosphorylation of 5'-hydroxyl termini in nucleic acids, which play a crucial role in DNA recombination, replication and damage repair. Here, a microchip electrophoresis laser induced fluorescence (MCE-LIF) method based on biochemical reaction was developed for the detection of T4 PNK activity and inhibitors. In this method, the single strand DNA (ssDNA) was hybridized with the 5-carboxyfluorescein (FAM) labeled single strand DNA (ssDNA-FAM) to form FAM labeled double-stranded DNA (dsDNA-FAM). In the presence of T4 PNK and adenosine triphosphate (ATP), T4 PNK catalyzes the transfer of γ-phosphate residues from ATP to the 5-hydroxyl terminal of dsDNA-FAM. The phosphorylated dsDNA-FAM can be gradually hydrolyzed by λexo to produce a FAM labeled single nucleotide fragment. Then the FAM labeled single nucleotide fragment and the unhydrolyzed dsDNA-FAM were separated by MCE, and two electrophoresis peaks appeared in the electrophoretogram. The detection of T4 PNK activity and inhibitors was realized by measuring the peak height of the FAM labeled single nucleotide fragment in electrophoretogram. This assay is very sensitive with a limit of detection of 0.002 U/mL, and it can be further used to screen the T4 PNK inhibitors.
Collapse
Affiliation(s)
- Yan Zhang
- State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin, 541004, China
| | - Jingjin Zhao
- State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin, 541004, China.
| | - Shenyu Chen
- State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin, 541004, China
| | - Shuting Li
- State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin, 541004, China
| | - Shulin Zhao
- State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin, 541004, China.
| |
Collapse
|
20
|
Advances in capillary electrophoresis for the life sciences. J Chromatogr B Analyt Technol Biomed Life Sci 2019; 1118-1119:116-136. [PMID: 31035134 DOI: 10.1016/j.jchromb.2019.04.020] [Citation(s) in RCA: 78] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2018] [Revised: 03/15/2019] [Accepted: 04/09/2019] [Indexed: 12/19/2022]
Abstract
Capillary electrophoresis (CE) played an important role in developments in the life sciences. The technique is nowadays used for the analysis of both large and small molecules in applications where it performs better than or is complementary to liquid chromatographic techniques. In this review, principles of different electromigration techniques, especially capillary isoelectric focusing (CIEF), capillary gel (CGE) and capillary zone electrophoresis (CZE), are described and recent developments in instrumentation, with an emphasis on mass spectrometry (MS) coupling and microchip CE, are discussed. The role of CE in the life sciences is shown with applications in which it had a high impact over the past few decades. In this context, current practice for the characterization of biopharmaceuticals (therapeutic proteins) is shown with CIEF, CGE and CZE using different detection techniques, including MS. Subsequently, the application of CGE and CZE, in combination with laser induced fluorescence detection and CZE-MS are demonstrated for the analysis of protein-released glycans in the characterization of biopharmaceuticals and glycan biomarker discovery in biological samples. Special attention is paid to developments in capillary coatings and derivatization strategies for glycans. Finally, routine CE analysis in clinical chemistry and latest developments in metabolomics approaches for the profiling of small molecules in biological samples are discussed. The large number of CE applications published for these topics in recent years clearly demonstrates the established role of CE in life sciences.
Collapse
|
21
|
He M, Luo P, Hong J, Wang X, Wu H, Zhang R, Qu F, Xiang Y, Xu W. Structural Analysis of Biomolecules through a Combination of Mobility Capillary Electrophoresis and Mass Spectrometry. ACS OMEGA 2019; 4:2377-2386. [PMID: 31459477 PMCID: PMC6648644 DOI: 10.1021/acsomega.8b03224] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/19/2018] [Accepted: 01/24/2019] [Indexed: 05/08/2023]
Abstract
The 3D structures of biomolecules determine their biological function. Established methods in biomolecule structure determination typically require purification, crystallization, or modification of target molecules, which limits their applications for analyzing trace amounts of biomolecules in complex matrices. Here, we developed instruments and methods of mobility capillary electrophoresis (MCE) and its coupling with MS for the 3D structural analysis of biomolecules in the liquid phase. Biomolecules in complex matrices could be separated by MCE and sequentially detected by MS. The effective radius and the aspect ratio of each separated biomolecule were simultaneously determined through the separation by MCE, which were then used as restraints in determining biomolecule conformations through modeling. Feasibility of this method was verified by analyzing a mixture of somatostatin and bradykinin, two peptides with known liquid-phase structures. Proteins could also be structurally analyzed using this method, which was demonstrated for lysozyme. The combination of MCE and MS for complex sample analysis was also demonstrated. MCE and MCE-MS would allow us to analyze trace amounts of biomolecules in complex matrices, which has the potential to be an alternative and powerful biomolecule structure analysis technique.
Collapse
Affiliation(s)
- Muyi He
- College
of Information Science, Shenzhen University, Shenzhen 518060, China
- School
of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Pan Luo
- School
of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Jie Hong
- School
of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Xiaofeng Wang
- Institute
of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China
| | - Haimei Wu
- School
of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Rongkai Zhang
- School
of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Feng Qu
- School
of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Ye Xiang
- Beijing
Advanced Innovation Center for Structural Biology, Department of Basic
Medical Sciences, School of Medicine, Tsinghua
University, Beijing 100084, China
- E-mail: (Y.X.)
| | - Wei Xu
- College
of Information Science, Shenzhen University, Shenzhen 518060, China
- School
of Life Science, Beijing Institute of Technology, Beijing 100081, China
- E-mail: (W.X.)
| |
Collapse
|
22
|
Peng W, Zhao J, Dong X, Banazadeh A, Huang Y, Hussien A, Mechref Y. Clinical application of quantitative glycomics. Expert Rev Proteomics 2018; 15:1007-1031. [PMID: 30380947 PMCID: PMC6647030 DOI: 10.1080/14789450.2018.1543594] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Aberrant glycosylation has been associated with many diseases. Decades of research activities have reported many reliable glycan biomarkers of different diseases which enable effective disease diagnostics and prognostics. However, none of the glycan markers have been approved for clinical diagnosis. Thus, a review of these studies is needed to guide the successful clinical translation. Area covered: In this review, we describe and discuss advances in analytical methods enabling clinical glycan biomarker discovery, focusing only on studies of released glycans. This review also summarizes the different glycobiomarkers identified for cancers, Alzheimer's disease, diabetes, hepatitis B and C, and other diseases. Expert commentary: Along with the development of techniques in quantitative glycomics, more glycans or glycan patterns have been reported as better potential biomarkers of different diseases and proved to have greater diagnostic/diagnostic sensitivity and specificity than existing markers. However, to successfully apply glycan markers in clinical diagnosis, more studies and verifications on large biological cohorts need to be performed. In addition, faster and more efficient glycomic strategies need to be developed to shorten the turnaround time. Thus, glycan biomarkers have an immense chance to be used in clinical prognosis and diagnosis of many diseases in the near future.
Collapse
Affiliation(s)
- Wenjing Peng
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, 79409, United States
| | - Jingfu Zhao
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, 79409, United States
| | - Xue Dong
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, 79409, United States
| | - Alireza Banazadeh
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, 79409, United States
| | - Yifan Huang
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, 79409, United States
| | - Ahmed Hussien
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, 79409, United States
- Department of Biotechnology, Institute of Graduate Studies and Research, University of Alexandria, Alexandria, 21526, Egypt
| | - Yehia Mechref
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, 79409, United States
| |
Collapse
|
23
|
Dong X, Huang Y, Cho BG, Zhong J, Gautam S, Peng W, Williamson SD, Banazadeh A, Torres-Ulloa KY, Mechref Y. Advances in mass spectrometry-based glycomics. Electrophoresis 2018; 39:3063-3081. [PMID: 30199110 DOI: 10.1002/elps.201800273] [Citation(s) in RCA: 72] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2018] [Revised: 09/05/2018] [Accepted: 09/06/2018] [Indexed: 12/22/2022]
Abstract
The diversification of the chemical properties and biological functions of proteins is attained through posttranslational modifications, such as glycosylation. Glycans, which are covalently attached to proteins, play a vital role in cell activities. The microheterogeneity and complexity of glycan structures associated with proteins make comprehensive glycomic analysis challenging. However, recent advancements in mass spectrometry (MS), separation techniques, and sample preparation methods have primarily facilitated structural elucidation and quantitation of glycans. This review focuses on describing recent advances in MS-based techniques used for glycomic analysis (2012-2018), including ionization, tandem MS, and separation techniques coupled with MS. Progress in glycomics workflow involving glycan release, purification, derivatization, and separation will also be highlighted here. Additionally, the recent development of quantitative glycomics through comparative and multiplex approaches will also be described.
Collapse
Affiliation(s)
- Xue Dong
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA
| | - Yifan Huang
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA
| | - Byeong Gwan Cho
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA
| | - Jieqiang Zhong
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA
| | - Sakshi Gautam
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA
| | - Wenjing Peng
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA
| | - Seth D Williamson
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA
| | - Alireza Banazadeh
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA
| | - Katya Y Torres-Ulloa
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA
| | - Yehia Mechref
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA
| |
Collapse
|
24
|
Lu G, Crihfield CL, Gattu S, Veltri LM, Holland LA. Capillary Electrophoresis Separations of Glycans. Chem Rev 2018; 118:7867-7885. [PMID: 29528644 PMCID: PMC6135675 DOI: 10.1021/acs.chemrev.7b00669] [Citation(s) in RCA: 112] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2017] [Indexed: 01/04/2023]
Abstract
Capillary electrophoresis has emerged as a powerful approach for carbohydrate analyses since 2014. The method provides high resolution capable of separating carbohydrates by charge-to-size ratio. Principle applications are heavily focused on N-glycans, which are highly relevant to biological therapeutics and biomarker research. Advances in techniques used for N-glycan structural identification include migration time indexing and exoglycosidase and lectin profiling, as well as mass spectrometry. Capillary electrophoresis methods have been developed that are capable of separating glycans with the same monosaccharide sequence but different positional isomers, as well as determining whether monosaccharides composing a glycan are alpha or beta linked. Significant applications of capillary electrophoresis to the analyses of N-glycans in biomarker discovery and biological therapeutics are emphasized with a brief discussion included on carbohydrate analyses of glycosaminoglycans and mono-, di-, and oligosaccharides relevant to food and plant products. Innovative, emerging techniques in the field are highlighted and the future direction of the technology is projected based on the significant contributions of capillary electrophoresis to glycoscience from 2014 to the present as discussed in this review.
Collapse
Affiliation(s)
- Grace Lu
- C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia 26506, United States
| | - Cassandra L. Crihfield
- C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia 26506, United States
| | - Srikanth Gattu
- C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia 26506, United States
| | - Lindsay M. Veltri
- C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia 26506, United States
| | - Lisa A. Holland
- C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia 26506, United States
| |
Collapse
|
25
|
Banazadeh A, Peng W, Veillon L, Mechref Y. Carbon Nanoparticles and Graphene Nanosheets as MALDI Matrices in Glycomics: a New Approach to Improve Glycan Profiling in Biological Samples. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2018; 29:1892-1900. [PMID: 29916086 PMCID: PMC6298861 DOI: 10.1007/s13361-018-1985-z] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/09/2018] [Revised: 04/28/2018] [Accepted: 04/28/2018] [Indexed: 05/15/2023]
Abstract
Glycomics continues to be a highly dynamic and interesting research area due to the need to comprehensively understand the biological attributes of glycosylation in many important biological functions such as the immune response, cell development, cell differentiation/adhesion, and host-pathogen interactions. Although matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has proven to be suitable for glycomic profiling studies, there is a need for improved sensitivity in the detection of native glycans, which ionize inefficiently. In this study, we investigated the efficiencies of graphene nanosheets (GNs) and carbon nanoparticles (CNPs) as MALDI matrices and co-matrices in glycan profiling. Our results indicated an enhancement of signal intensity by several orders of magnitude upon using GNs and CNPs in MALDI analysis of N-glycans derived from a variety of biological samples. Interestingly, increasing the amounts of CNPs and GNs improved not only the signal intensities but also prompted in-source decay (ISD) fragmentations, which produced extensive glycosidic and cross-ring cleavages. Our results indicated that the extent of ISD fragmentation could be modulated by CNP and GN concentrations, to obtain MS2 and pseudo-MS3 spectra. The results for glycan profiling in high salt solutions confirmed high salt-tolerance capacities for both CNPs and GNs. Finally, the results showed that by using CNPs and GNs as co-matrices, DHB crystal formation was more homogeneous which improved shot-to-shot reproducibility and sensitivity. Graphical Abstract ᅟ.
Collapse
Affiliation(s)
- Alireza Banazadeh
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409-1061, USA
| | - Wenjing Peng
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409-1061, USA
| | - Lucas Veillon
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409-1061, USA
| | - Yehia Mechref
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409-1061, USA.
| |
Collapse
|
26
|
Greco V, Piras C, Pieroni L, Ronci M, Putignani L, Roncada P, Urbani A. Applications of MALDI-TOF mass spectrometry in clinical proteomics. Expert Rev Proteomics 2018; 15:683-696. [PMID: 30058389 DOI: 10.1080/14789450.2018.1505510] [Citation(s) in RCA: 49] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
INTRODUCTION The development of precision medicine requires advanced technologies to address the multifactorial disease stratification and to support personalized treatments. Among omics techniques, proteomics based on Mass Spectrometry (MS) is becoming increasingly relevant in clinical practice allowing a phenotypic characterization of the dynamic functional status of the organism. From this perspective, Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) MS is a suitable platform for providing a high-throughput support to clinics. Areas covered: This review aims to provide an updated overview of MALDI-TOF MS applications in clinical proteomics. The most relevant features of this analysis have been discussed, highlighting both pre-analytical and analytical factors that are crucial in proteomics studies. Particular emphasis is placed on biofluids proteomics for biomarkers discovery and on recent progresses in clinical microbiology, drug monitoring, and minimal residual disease (MRD). Expert commentary: Despite some analytical limitations, the latest technological advances together with the easiness of use, the low time and low cost consuming and the high throughput are making MALDI-TOF MS instruments very attractive for the clinical practice. These features offer a significant potential for the routine of the clinical laboratory and ultimately for personalized medicine.
Collapse
Affiliation(s)
- Viviana Greco
- a Institute of Biochemistry and Clinical Biochemistry , Università Cattolica del Sacro Cuore , Rome , Italy.,b Department of Laboratory Diagnostic and Infectious Diseases , Fondazione Policlinico Universitario Agostino Gemelli-IRCCS , Rome , Italy
| | - Cristian Piras
- c Dipartimento di Medicina Veterinaria , Università degli studi di Milano , Milano , Italy
| | - Luisa Pieroni
- d Proteomics and Metabonomics Unit , IRCCS-Fondazione Santa Lucia , Rome , Italy
| | - Maurizio Ronci
- d Proteomics and Metabonomics Unit , IRCCS-Fondazione Santa Lucia , Rome , Italy.,e Department of Medical, Oral and Biotechnological Sciences , University "G. D'Annunzio" of Chieti-Pescara , Chieti , Italy
| | - Lorenza Putignani
- f Unit of Parasitology Bambino Gesù Children's Hospital , IRCCS , Rome , Italy.,g Unit of Human Microbiome , Bambino Gesù Children's Hospital, IRCCS , Rome , Italy
| | - Paola Roncada
- h Dipartimento di Scienze della Salute , Università degli studi "Magna Græcia" di Catanzaro , Catanzaro , Italy
| | - Andrea Urbani
- a Institute of Biochemistry and Clinical Biochemistry , Università Cattolica del Sacro Cuore , Rome , Italy.,b Department of Laboratory Diagnostic and Infectious Diseases , Fondazione Policlinico Universitario Agostino Gemelli-IRCCS , Rome , Italy
| |
Collapse
|
27
|
Wuethrich A, Quirino JP. A decade of microchip electrophoresis for clinical diagnostics - A review of 2008-2017. Anal Chim Acta 2018; 1045:42-66. [PMID: 30454573 DOI: 10.1016/j.aca.2018.08.009] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2018] [Revised: 07/30/2018] [Accepted: 08/03/2018] [Indexed: 01/10/2023]
Abstract
A core element in clinical diagnostics is the data interpretation obtained through the analysis of patient samples. To obtain relevant and reliable information, a methodological approach of sample preparation, separation, and detection is required. Traditionally, these steps are performed independently and stepwise. Microchip capillary electrophoresis (MCE) can provide rapid and high-resolution separation with the capability to integrate a streamlined and complete diagnostic workflow suitable for the point-of-care setting. Whilst standard clinical diagnostics methods normally require hours to days to retrieve specific patient data, MCE can reduce the time to minutes, hastening the delivery of treatment options for the patients. This review covers the advances in MCE for disease detection from 2008 to 2017. Miniaturised diagnostic approaches that required an electrophoretic separation step prior to the detection of the biological samples are reviewed. In the two main sections, the discussion is focused on the technical set-up used to suit MCE for disease detection and on the strategies that have been applied to study various diseases. Throughout these discussions MCE is compared to other techniques to create context of the potential and challenges of MCE. A comprehensive table categorised based on the studied disease using MCE is provided. We also comment on future challenges that remain to be addressed.
Collapse
Affiliation(s)
- Alain Wuethrich
- Centre for Personalised Nanomedicine, Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland, Building 75, Brisbane, QLD, 4072, Australia
| | - Joselito P Quirino
- Australian Centre for Research on Separation Science (ACROSS), School of Physical Sciences-Chemistry, University of Tasmania, Private Bag 75, Hobart, TAS, 7001, Australia.
| |
Collapse
|
28
|
Lee PY, Chin SF, Low TY, Jamal R. Probing the colorectal cancer proteome for biomarkers: Current status and perspectives. J Proteomics 2018; 187:93-105. [PMID: 29953962 DOI: 10.1016/j.jprot.2018.06.014] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2018] [Revised: 06/13/2018] [Accepted: 06/23/2018] [Indexed: 02/07/2023]
Abstract
Colorectal cancer (CRC) is one of the most prevalent malignancies worldwide. Biomarkers that can facilitate better clinical management of CRC are in high demand to improve patient outcome and to reduce mortality. In this regard, proteomic analysis holds a promising prospect in the hunt of novel biomarkers for CRC and in understanding the mechanisms underlying tumorigenesis. This review aims to provide an overview of the current progress of proteomic research, focusing on discovery and validation of diagnostic biomarkers for CRC. We will summarize the contributions of proteomic strategies to recent discoveries of protein biomarkers for CRC and also briefly discuss the potential and challenges of different proteomic approaches in biomarker discovery and translational applications.
Collapse
Affiliation(s)
- Pey Yee Lee
- UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia.
| | - Siok-Fong Chin
- UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia
| | - Teck Yew Low
- UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia
| | - Rahman Jamal
- UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia
| |
Collapse
|
29
|
Benktander JD, Gizaw ST, Gaunitz S, Novotny MV. Analytical Scheme Leading to Integrated High-Sensitivity Profiling of Glycosphingolipids Together with N- and O-Glycans from One Sample. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2018; 29:1125-1137. [PMID: 29744812 PMCID: PMC6226365 DOI: 10.1007/s13361-018-1933-y] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/29/2017] [Revised: 02/23/2018] [Accepted: 02/23/2018] [Indexed: 05/15/2023]
Abstract
Glycoconjugates are directly or indirectly involved in many biological processes. Due to their complex structures, the structural elucidation of glycans and the exploration of their role in biological systems have been challenging. Glycan pools generated through release from glycoprotein or glycolipid mixtures can often be very complex. For the sake of procedural simplicity, many glycan profiling studies choose to concentrate on a single class of glycoconjugates. In this paper, we demonstrate it feasible to cover glycosphingolipids, N-glycans, and O-glycans isolated from the same sample. Small volumes of human blood serum and ascites fluid as well as small mouse brain tissue samples are sufficient to profile sequentially glycans from all three classes of glycoconjugates and even positively identify some mixture components through MALDI-MS and LC-ESI-MS. The results show that comprehensive glycan profiles can be obtained from the equivalent of 500-μg protein starting material or possibly less. These methodological improvements can help accelerating future glycomic comprehensive studies, especially for precious clinical samples. Graphical Abstract Outline of glycan profiling procedures.
Collapse
Affiliation(s)
- John D Benktander
- Department of Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN, 47405, USA
| | - Solomon T Gizaw
- Department of Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN, 47405, USA
| | - Stefan Gaunitz
- Department of Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN, 47405, USA
| | - Milos V Novotny
- Department of Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN, 47405, USA.
| |
Collapse
|
30
|
Liu S, Cheng L, Fu Y, Liu BF, Liu X. Characterization of IgG N-glycome profile in colorectal cancer progression by MALDI-TOF-MS. J Proteomics 2018; 181:225-237. [DOI: 10.1016/j.jprot.2018.04.026] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2017] [Revised: 03/21/2018] [Accepted: 04/18/2018] [Indexed: 12/17/2022]
|
31
|
Rehulka P, Zahradnikova M, Rehulkova H, Dvorakova P, Nenutil R, Valik D, Vojtesek B, Hernychova L, Novotny MV. Microgradient separation technique for purification and fractionation of permethylated N-glycans before mass spectrometric analyses. J Sep Sci 2018; 41:1973-1982. [DOI: 10.1002/jssc.201701339] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2017] [Revised: 01/17/2018] [Accepted: 01/23/2018] [Indexed: 12/16/2022]
Affiliation(s)
- Pavel Rehulka
- Department of Molecular Pathology and Biology; Faculty of Military Health Sciences; University of Defence; Hradec Kralove Czech Republic
| | - Martina Zahradnikova
- Regional Centre for Applied Molecular Oncology; Masaryk Memorial Cancer Institute; Brno Czech Republic
| | - Helena Rehulkova
- Department of Molecular Pathology and Biology; Faculty of Military Health Sciences; University of Defence; Hradec Kralove Czech Republic
| | - Petra Dvorakova
- Regional Centre for Applied Molecular Oncology; Masaryk Memorial Cancer Institute; Brno Czech Republic
| | - Rudolf Nenutil
- Regional Centre for Applied Molecular Oncology; Masaryk Memorial Cancer Institute; Brno Czech Republic
| | - Dalibor Valik
- Regional Centre for Applied Molecular Oncology; Masaryk Memorial Cancer Institute; Brno Czech Republic
| | - Borivoj Vojtesek
- Regional Centre for Applied Molecular Oncology; Masaryk Memorial Cancer Institute; Brno Czech Republic
| | - Lenka Hernychova
- Regional Centre for Applied Molecular Oncology; Masaryk Memorial Cancer Institute; Brno Czech Republic
| | - Milos V. Novotny
- Regional Centre for Applied Molecular Oncology; Masaryk Memorial Cancer Institute; Brno Czech Republic
- Department of Chemistry; Indiana University; Bloomington IN USA
| |
Collapse
|
32
|
Kailemia MJ, Xu G, Wong M, Li Q, Goonatilleke E, Leon F, Lebrilla CB. Recent Advances in the Mass Spectrometry Methods for Glycomics and Cancer. Anal Chem 2018; 90:208-224. [PMID: 29049885 PMCID: PMC6200424 DOI: 10.1021/acs.analchem.7b04202] [Citation(s) in RCA: 61] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Affiliation(s)
- Muchena J. Kailemia
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
- These authors contributed equally to this work
| | - Gege Xu
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
- These authors contributed equally to this work
| | - Maurice Wong
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
| | - Qiongyu Li
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
| | - Elisha Goonatilleke
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
| | - Frank Leon
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
| | - Carlito B. Lebrilla
- Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, CA 95616, United States
- Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA
- Foods for Health Institute, University of California, Davis, CA 95616, USA
| |
Collapse
|
33
|
Distinguishing glycan isomers by voltammetry. Modification of 2,3-sialyllactose and 2,6-sialyllactose by osmium(VI) complexes. Electrochem commun 2017. [DOI: 10.1016/j.elecom.2017.10.014] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
|
34
|
Novotny MV. Development of capillary liquid chromatography: A personal perspective. J Chromatogr A 2017; 1523:3-16. [PMID: 28701267 PMCID: PMC5675780 DOI: 10.1016/j.chroma.2017.06.042] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2017] [Revised: 06/13/2017] [Accepted: 06/15/2017] [Indexed: 11/21/2022]
Abstract
This is a historical account on the development of capillary LC from its beginning to the present day. The first investigations into the viability of capillary LC date back to the late 1970s, a decade after the pioneering efforts in HPLC. The drastically reduced column dimensions were required to counter the slow solute diffusion in liquids. There were numerous instrumental difficulties with sample introduction and detection in the picoliter or even femtoliter volumes. High-efficiency separations were needed in the analysis of complex biological mixtures. Miniaturization brought distinct advantages in spectroscopic and electrochemical detection. Since the 1980s, column technologies underwent significant changes: (a) from glass-drawn microcapillaries to slurry-packed, small-diameter fused silica columns; and (b) in microcapillaries packed alternatively with sub-2-μm particles or monoliths. The viability of LC-MS combination has dramatically promoted the use of small-diameter capillaries. Through "omics technologies", capillary LC/tandem MS accounts for most applications in proteomics, glycomics and metabolomics.
Collapse
Affiliation(s)
- Milos V Novotny
- Department of Chemistry, Indiana University, 800 E. Kirkwood Ave., Bloomington, IN 47405, USA.
| |
Collapse
|
35
|
Snyder CM, Zhou X, Karty JA, Fonslow BR, Novotny MV, Jacobson SC. Capillary electrophoresis-mass spectrometry for direct structural identification of serum N-glycans. J Chromatogr A 2017; 1523:127-139. [PMID: 28989033 DOI: 10.1016/j.chroma.2017.09.009] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2017] [Revised: 09/01/2017] [Accepted: 09/03/2017] [Indexed: 10/18/2022]
Abstract
Through direct coupling of capillary electrophoresis (CE) to mass spectrometry (MS) with a sheathless interface, we have identified 77 potential N-glycan structures derived from human serum. We confirmed the presence of N-glycans previously identified by indirect methods, e.g., electrophoretic mobility standards, obtained 31 new N-glycan structures not identified in our prior work, differentiated co-migrating structures, and determined specific linkages on isomers featuring sialic acids. Serum N-glycans were cleaved from proteins, neutralized via methylamidation, and labeled with the fluorescent tag 8-aminopyrene-1,3,6-trisulfonic acid, which renders the glycan fluorescent and provides a -3 charge for electrophoresis and negative-mode MS detection. The neutralization reaction also stabilizes the labile sialic acids. In addition to methylamidation, native charges from sialic acids were neutralized through reaction with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium to amidate α2,6-linked sialic acids in the presence of ammonium chloride and form lactones with α2,3-linked sialic acids. This neutralization effectively labels each type of sialic acid with a unique mass to determine specific linkages on sialylated N-glycans. For both neutralization schemes, we compared the results from microchip electrophoresis and CE.
Collapse
Affiliation(s)
- Christa M Snyder
- Department of Chemistry, Indiana University, Bloomington, IN 47405-7102, United States
| | - Xiaomei Zhou
- Department of Chemistry, Indiana University, Bloomington, IN 47405-7102, United States
| | - Jonathan A Karty
- Department of Chemistry, Indiana University, Bloomington, IN 47405-7102, United States
| | | | - Milos V Novotny
- Department of Chemistry, Indiana University, Bloomington, IN 47405-7102, United States
| | - Stephen C Jacobson
- Department of Chemistry, Indiana University, Bloomington, IN 47405-7102, United States.
| |
Collapse
|
36
|
Wang B, Akiba U, Anzai JI. Recent Progress in Nanomaterial-Based Electrochemical Biosensors for Cancer Biomarkers: A Review. Molecules 2017; 22:E1048. [PMID: 28672780 PMCID: PMC6152304 DOI: 10.3390/molecules22071048] [Citation(s) in RCA: 59] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2017] [Revised: 06/21/2017] [Accepted: 06/22/2017] [Indexed: 02/07/2023] Open
Abstract
This article reviews recent progress in the development of nanomaterial-based electrochemical biosensors for cancer biomarkers. Because of their high electrical conductivity, high affinity to biomolecules, and high surface area-to-weight ratios, nanomaterials, including metal nanoparticles, carbon nanotubes, and graphene, have been used for fabricating electrochemical biosensors. Electrodes are often coated with nanomaterials to increase the effective surface area of the electrodes and immobilize a large number of biomolecules such as enzymes and antibodies. Alternatively, nanomaterials are used as signaling labels for increasing the output signals of cancer biomarker sensors, in which nanomaterials are conjugated with secondary antibodies and redox compounds. According to this strategy, a variety of biosensors have been developed for detecting cancer biomarkers. Recent studies show that using nanomaterials is highly advantageous in preparing high-performance biosensors for detecting lower levels of cancer biomarkers. This review focuses mainly on the protocols for using nanomaterials to construct cancer biomarker sensors and the performance characteristics of the sensors. Recent trends in the development of cancer biomarker sensors are discussed according to the nanomaterials used.
Collapse
Affiliation(s)
- Baozhen Wang
- Department of Nutrition and Food Hygiene, School of Public Health, Shandong University, 44 Wenhua Xilu, Jinan 250012, China.
| | - Uichi Akiba
- Graduate School of Engineering and Science, Akita University, 1-1 Tegatagakuen-machi, Akita 010-8502, Japan.
| | - Jun-Ichi Anzai
- Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
| |
Collapse
|
37
|
Holland LA, Gattu S, Crihfield CL, Bwanali L. Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of β(1-3)-linked galactose in glycans. J Chromatogr A 2017. [PMID: 28647147 DOI: 10.1016/j.chroma.2017.06.038] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
A thermally responsive nanogel is used to create stationary zones of enzyme and lectin in a separation capillary. Once patterned in the capillary, analyte is driven through the zone, where it is converted to a specific product if an enzyme is used or captured if a lectin is used. These stationary zones are easily expelled after the analysis and then re-patterned in the capillary. The nanogel is compatible with enzymes and lectins and improves the stability of galactosidase, enabling more cost-effective use of biological reagents that provide insight into glycan structure. A feature of using stationary zones is that the reaction time can be controlled by the length of the zone, the applied field controlling the analyte mobility, or the use of electrophoretic mixing by switching the polarity of the applied voltage while the analyte is located in the zone. The temperature, applied voltage, and length of the stationary zone, which are factors that enhance the performance of the enzyme, are characterized. The combined use of enzymes and lectins in capillary electrophoresis is a new strategy to advance rapid and automated analyses of glycans using nanoliter volumes of enzymes and lectins. The applicability of this use of stationary zones of enzyme and lectin in capillary electrophoresis is demonstrated with the identification of β(1-3)-linked galactose in N-glycan.
Collapse
Affiliation(s)
- Lisa A Holland
- C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506, United States.
| | - Srikanth Gattu
- C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506, United States
| | - Cassandra L Crihfield
- C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506, United States
| | - Lloyd Bwanali
- C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506, United States
| |
Collapse
|
38
|
Zou G, Benktander JD, Gizaw ST, Gaunitz S, Novotny MV. Comprehensive Analytical Approach toward Glycomic Characterization and Profiling in Urinary Exosomes. Anal Chem 2017; 89:5364-5372. [PMID: 28402650 DOI: 10.1021/acs.analchem.7b00062] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Exosomes are extracellular nanosized vesicles with lipid bilayers encapsulating nucleic acids and proteins, both with and without glycosylation. While exosomal nucleic acids and proteins have previously been explored to identify cancer biomarkers with some promising results, little information has been available concerning their glycoconjugate content. Exosomes were isolated from normal urine samples through multistep differential centrifugation. The isolated exosomes have an average size of 146 nm and a spherical shape, as determined by dynamic light scattering and transmission electron microscopy, respectively. N-Glycans were enzymatically released from the isolated vesicles. After being reduced and permethylated, N-glycans were measured by MALDI mass spectrometry. Paucimannosidic, high-mannose, and complex type glycans were identified and their relative abundances were determined. Some detailed structures of these glycans were revealed through liquid chromatography/tandem mass spectrometry (LC/MS-MS). The reduced N-glycans, without being permethylated, were also separated and analyzed by LC/MS-MS, and their structures were further detailed through isomeric separation on porous graphitized carbon (PGC) packed in long capillaries. Using microfractionation before LC/MS-MS, minor multiantennary N-glycans were preconcentrated as based on hydrophobicity or charge. Preconcentration of the reduced and permethylated glycans on a C18 cartridge revealed numerous large glycans, whereas fractionation of the reduced N-glycans by ion-exchange cartridges facilitated detection of sulfated glycans. After removing N-glycans from the original sample aliquot, O-glycans were chemically released from urinary exosomes and profiled, revealing some unusual structures.
Collapse
Affiliation(s)
- Guozhang Zou
- Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States
| | - John D Benktander
- Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States
| | - Solomon T Gizaw
- Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States
| | - Stefan Gaunitz
- Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States
| | - Milos V Novotny
- Department of Chemistry, Indiana University , Bloomington, Indiana 47405, United States
| |
Collapse
|
39
|
Affiliation(s)
- Stefan Gaunitz
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States
| | - Gabe Nagy
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States
| | - Nicola L. B. Pohl
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States
| | - Milos V. Novotny
- Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States
- Regional Center for Applied Molecular Oncology, Masaryk Memorial Oncological Institute, 656 53 Brno, Czech Republic
| |
Collapse
|