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1
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Šoić D, Kifer D, Szavits-Nossan J, Blivajs A, Đerek L, Rudan D, Gornik O, Gudelj I, Keser T. High-Throughput Site-Specific N-Glycosylation Profiling of Human Fibrinogen in Atrial Fibrillation. J Proteome Res 2025; 24:2121-2134. [PMID: 40099449 PMCID: PMC11976851 DOI: 10.1021/acs.jproteome.5c00096] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2025] [Revised: 03/03/2025] [Accepted: 03/07/2025] [Indexed: 03/19/2025]
Abstract
Fibrinogen is a major plasma glycoprotein involved in blood coagulation and inflammatory responses. Alterations in its glycosylation have been implicated in various pathological conditions; yet, its site-specific N-glycosylation profile remains largely unexplored in a clinical context. Here, we present a high-throughput LC-MS workflow for site-specific analysis of fibrinogen N-glycosylation using a cost-effective ethanol precipitation enrichment method. The method demonstrated good intra- and interplate repeatability (CV: 5% and 12%, respectively) and was validated through the first assessment of intraindividual temporal stability in healthy individuals, revealing consistent glycosylation patterns within individuals. Application to 181 atrial fibrillation (AF) patients and 52 healthy controls identified three gamma chain glycoforms significantly associated with AF. Most notably, increased levels of the asialylated N4H5, known to enhance fibrin bundle thickness and promote clot formation, suggest a potential mechanism linking glycosylation changes to the prothrombotic state in AF. Furthermore, fibrinogen sialylation showed strong associations with cardiovascular risk factors, including triglycerides, BMI, and glucose levels. Longitudinal analysis of 108 AF patients six months postcatheter ablation showed stability in the AF-associated glycan profile. Our findings establish fibrinogen glycosylation as a potential biomarker for cardiovascular conditions and demonstrate the utility of site-specific glycosylation analysis for clinical applications.
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Affiliation(s)
- Dinko Šoić
- Faculty
of Pharmacy and Biochemistry, University
of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia
| | - Domagoj Kifer
- Faculty
of Pharmacy and Biochemistry, University
of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia
| | - Janko Szavits-Nossan
- Magdalena
University Hospital for Cardiovascular Diseases, Radnička cesta 32, 10000 Zagreb, Croatia
- Faculty
of Dental Medicine and Health, J.J. Strossmayer
University in Osijek, Crkvena 21, 31000 Osijek, Croatia
- Faculty
of Medicine, J.J. Strossmayer University
of Osijek, Josipa Huttlera
4, 31000 Osijek, Croatia
| | - Aleksandar Blivajs
- Department
of Cardiology, University Hospital Dubrava, Avenija Gojka Šuška
6, 10000 Zagreb, Croatia
| | - Lovorka Đerek
- Clinical
Department for Laboratory Diagnostics, University
Hospital Dubrava, Avenija
Gojka Šuška 6, 10000 Zagreb, Croatia
| | - Diana Rudan
- Department
of Cardiology, University Hospital Dubrava, Avenija Gojka Šuška
6, 10000 Zagreb, Croatia
| | - Olga Gornik
- Faculty
of Pharmacy and Biochemistry, University
of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia
| | - Ivan Gudelj
- Faculty
of Biotechnology and Drug Development, University
of Rijeka, Radmile Matejčić 2, 51000 Rijeka, Croatia
| | - Toma Keser
- Faculty
of Pharmacy and Biochemistry, University
of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia
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2
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Kalidas N, Peddada N, Pandey K, Ashish. SAXS data based glycosylated models of human alpha-1-acid glycorprotein, a key player in health, disease and drug circulation. J Biomol Struct Dyn 2025:1-15. [PMID: 40056387 DOI: 10.1080/07391102.2025.2475244] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2024] [Accepted: 02/02/2025] [Indexed: 03/10/2025]
Abstract
Plasma Alpha-1-glycoprotein (AGP) binds diverse drugs, its isoforms and their levels vary significantly in acute phases of health. Relative binding pattern of drugs to AGP and albumin has been used to model their release profiles, and structural insights on glycosylated form of AGP will improve predictions. Main challenge is the heavy and heterogeneous glycosylation of AGP molecules. Our small angle X-ray scattering (SAXS) data on plasma extracted AGP showed interparticulate effect from 283 to 313 K which disappeared irreversibly upon further heating to 343K. Using ALPHAFOLD2 server, the protein only portion could be modelled but as expected its theoretical SAXS profile did not match acquired experimental data. Using mass spectra-based information, we attached representative glycan motifs at known sites to compute four models of fully glycosylated AGP. Importantly, calculated SAXS profiles of these models agreed with our experimental data. These representative glycosylated models were further analyzed for molecular motions using Normal Mode Analysis and all-atom Molecular Dynamics simulations in reference to SAXS data. Overall, we show that SAXS data-based models of glycoprotein are better representation of this biopharmaceutical molecule and provide them for structure-based drug profile estimations.
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Affiliation(s)
- Nidhi Kalidas
- CSIR-Institute of Microbial Technology, Chandigarh, India
| | - Nagesh Peddada
- CSIR-Institute of Microbial Technology, Chandigarh, India
| | - Kalpana Pandey
- CSIR-Institute of Microbial Technology, Chandigarh, India
| | - Ashish
- CSIR-Institute of Microbial Technology, Chandigarh, India
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3
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Nagai-Okatani C, Tominaga D, Tomioka A, Sakaue H, Goda N, Ko S, Kuno A, Kaji H. GRable Version 1.0: A Software Tool for Site-Specific Glycoform Analysis With Improved MS1-Based Glycopeptide Detection With Parallel Clustering and Confidence Evaluation With MS2 Information. Mol Cell Proteomics 2024; 23:100833. [PMID: 39181535 PMCID: PMC11421343 DOI: 10.1016/j.mcpro.2024.100833] [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: 11/02/2023] [Revised: 08/16/2024] [Accepted: 08/19/2024] [Indexed: 08/27/2024] Open
Abstract
High-throughput intact glycopeptide analysis is crucial for elucidating the physiological and pathological status of the glycans attached to each glycoprotein. Mass spectrometry-based glycoproteomic methods are challenging because of the diversity and heterogeneity of glycan structures. Therefore, we developed an MS1-based site-specific glycoform analysis method named "Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile (Glyco-RIDGE)" for a more comprehensive analysis. This method detects glycopeptide signals as a cluster based on the mass and chromatographic properties of glycopeptides and then searches for each combination of core peptides and glycan compositions by matching their mass and retention time differences. Here, we developed a novel browser-based software named GRable for semi-automated Glyco-RIDGE analysis with significant improvements in glycopeptide detection algorithms, including "parallel clustering." This unique function improved the comprehensiveness of glycopeptide detection and allowed the analysis to focus on specific glycan structures, such as pauci-mannose. The other notable improvement is evaluating the "confidence level" of the GRable results, especially using MS2 information. This function facilitated reduced misassignment of the core peptide and glycan composition and improved the interpretation of the results. Additional improved points of the algorithms are "correction function" for accurate monoisotopic peak picking; one-to-one correspondence of clusters and core peptides even for multiply sialylated glycopeptides; and "inter-cluster analysis" function for understanding the reason for detected but unmatched clusters. The significance of these improvements was demonstrated using purified and crude glycoprotein samples, showing that GRable allowed site-specific glycoform analysis of intact sialylated glycoproteins on a large-scale and in-depth. Therefore, this software will help us analyze the status and changes in glycans to obtain biological and clinical insights into protein glycosylation by complementing the comprehensiveness of MS2-based glycoproteomics. GRable can be freely run online using a web browser via the GlyCosmos Portal (https://glycosmos.org/grable).
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Affiliation(s)
- Chiaki Nagai-Okatani
- Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
| | - Daisuke Tominaga
- Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
| | - Azusa Tomioka
- Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
| | - Hiroaki Sakaue
- Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
| | - Norio Goda
- Department of Systems Medicine, Keio University School of Medicine, Shinjuku, Tokyo, Japan
| | - Shigeru Ko
- Department of Systems Medicine, Keio University School of Medicine, Shinjuku, Tokyo, Japan
| | - Atsushi Kuno
- Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
| | - Hiroyuki Kaji
- Molecular and Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan; Institute for Glyco-core Research (iGCORE), Nagoya University, Nagoya, Aichi, Japan.
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Ibrahim MA, Isah MB, Inim MD, Abdullahi AD, Adamu A. The connections of sialic acids and diabetes mellitus: therapeutic or diagnostic value? Glycobiology 2024; 34:cwae053. [PMID: 39041707 DOI: 10.1093/glycob/cwae053] [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: 03/11/2024] [Revised: 06/16/2024] [Accepted: 07/19/2024] [Indexed: 07/24/2024] Open
Abstract
Modulation of sialic acids is one of the important pathological consequences of both type 1 and type 2 diabetes mellitus with or without the micro- and macrovascular complications. However, the mechanistic, therapeutic and/or diagnostic implications of these observations are uncoordinated and possibly conflicting. This review critically analyses the scientific investigations connecting sialic acids with diabetes mellitus. Generally, variations in the levels and patterns of sialylation, fucosylation and galactosylation were predominant across various tissues and body systems of diabetic patients, but the immune system seemed to be most affected. These might be explored as a basis for differential diagnosis of various diabetic complications. Sialic acids are predominantly elevated in nearly all forms of diabetic conditions, particularly nephropathy and retinopathy, which suggests some diagnostic value but the mechanistic details were not unequivocal from the available data. The plausible mechanistic explanations for the elevated sialic acids are increased desialylation by sialidases, stimulation of hexosamine pathway and synthesis of acute phase proteins as well as oxidative stress. Additionally, sialic acids are also profoundly associated with glucose transport and insulin resistance in human-based studies while animal-based studies revealed that the increased desialylation of insulin receptors by sialidases, especially NEU1, might be the causal link. Interestingly, inhibition of the diabetes-associated NEU1 desialylation was beneficial in diabetes management and might be considered as a therapeutic target. It is hoped that the article will provide an informed basis for future research activities on the exploitation of sialic acids and glycobiology for therapeutic and/or diagnostic purposes against diabetes mellitus.
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Affiliation(s)
| | - Murtala Bindawa Isah
- Department of Biochemistry, Umaru Musa Yar'adua University, P.M.B. 2218, Katsina, Nigeria
| | - Mayen David Inim
- Department of Biochemistry, Ahmadu Bello University, Samaru, 80001, Zaria, Nigeria
| | | | - Auwal Adamu
- Department of Biochemistry, Ahmadu Bello University, Samaru, 80001, Zaria, Nigeria
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5
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van Schaick G, Wuhrer M, Blöchl C, Dolhain RJEM, Domínguez-Vega E. Anion Exchange Chromatography-Mass Spectrometry to Characterize Proteoforms of Alpha-1-Acid Glycoprotein during and after Pregnancy. J Proteome Res 2024; 23:2431-2440. [PMID: 38965920 PMCID: PMC11232096 DOI: 10.1021/acs.jproteome.4c00107] [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] [Indexed: 07/06/2024]
Abstract
Alpha-1-acid glycoprotein (AGP) is a heterogeneous glycoprotein fulfilling key roles in many biological processes, including transport of drugs and hormones and modulation of inflammatory and immune responses. The glycoform profile of AGP is known to change depending on (patho)physiological states such as inflammatory diseases or pregnancy. Besides complexity originating from five N-glycosylation sites, the heterogeneity of the AGP further expands to genetic variants. To allow in-depth characterization of this intriguing protein, we developed a method using anion exchange chromatography (AEX) coupled to mass spectrometry (MS) revealing the presence of over 400 proteoforms differing in their glycosylation or genetic variants. More precisely, we could determine that AGP mainly consists of highly sialylated higher antennary structures with on average 16 sialic acids and 0 or 1 fucose per protein. Interestingly, a slightly higher level of fucosylation was observed for AGP1 variants compared to that of AGP2. Proteoform assignment was supported by integrating data from complementary MS-based approaches, including AEX-MS of an exoglycosidase-treated sample and glycopeptide analysis after tryptic digestion. The developed analytical method was applied to characterize AGP from plasma of women during and after pregnancy, revealing differences in glycosylation profiles, specifically in the number of antennae, HexHexNAc units, and sialic acids.
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Affiliation(s)
- Guusje van Schaick
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
| | - Manfred Wuhrer
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
| | - Constantin Blöchl
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
| | - Radboud J E M Dolhain
- Department of Rheumatology, Erasmus Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands
| | - Elena Domínguez-Vega
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
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6
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Cindrić A, Pribić T, Lauc G. High-throughput N-glycan analysis in aging and inflammaging: State of the art and future directions. Semin Immunol 2024; 73:101890. [PMID: 39383621 DOI: 10.1016/j.smim.2024.101890] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/23/2024] [Revised: 10/01/2024] [Accepted: 10/02/2024] [Indexed: 10/11/2024]
Abstract
As the global population ages at an unprecedented rate, the prevalence of age-related diseases is increasing, making inflammaging - a phenomenon characterized by a chronic, low-grade inflammatory state that follows aging - a significant concern. Understanding the mechanisms of inflammaging and its impact on health is critical for developing strategies to improve the quality of life and manage health in the aging population. Despite their crucial roles in various biological processes, including immune response modulation, N-glycans, oligosaccharides covalently attached to many proteins, are often overlooked in clinical and research studies. This repeated oversight is largely due to their inherent complexity and the complexity of the analysis methods. High-throughput N-glycan analysis has emerged as a transformative tool in N-glycosylation research, enabling cost- and time-effective, detailed, and large-scale examination of N-glycan profiles. This paper is the first to explore the application of high-throughput N-glycomics techniques to investigate the complex interplay between N-glycosylation and the immune system in aging. Technological advancements have significantly improved Nglycan detection and characterization, providing insights into age-related changes in Nglycosylation. Key findings highlight consistent shifts in immunoglobulin G (IgG) and plasma/serum glycoprotein glycosylation with age, with a pronounced rise in agalactosylated structures bound to IgG that also affect the composition of the total plasma N-glycome. These N-glycan modifications seem to be strongly associated with inflammaging and have been identified as valuable biomarkers for biological age, predictors of disease risk, and proxy biomarkers for monitoring intervention efficacy at the individual level. Despite current challenges related to data complexity and methodological limitations, ongoing technological innovations and interdisciplinary research are expected tofurther advance our knowledge of glycan biology, improve diagnostic and therapeutic strategies, and promote healthier aging. The integration of glycomics with other omics approaches holds promise for a more comprehensive understanding of the aging immune system, paving the way for personalized medicine and targeted interventions to mitigate inflammaging. In conclusion, this paper underscores the transformative impact of high-throughput Nglycan analysis in aging and inflammaging.
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Affiliation(s)
- A Cindrić
- Genos Glycoscience Research Laboratory, Zagreb, Croatia
| | - T Pribić
- Genos Glycoscience Research Laboratory, Zagreb, Croatia
| | - G Lauc
- Genos Glycoscience Research Laboratory, Zagreb, Croatia; Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia.
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7
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Vučković F, Novokmet M, Šoić D, Štambuk J, Kolčić I, Polašek O, Lauc G, Gornik O, Keser T. Variability of human Alpha-1-acid glycoprotein N-glycome in a Caucasian population. Glycobiology 2024; 34:cwae031. [PMID: 38591797 DOI: 10.1093/glycob/cwae031] [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: 08/21/2023] [Revised: 04/04/2024] [Accepted: 04/04/2024] [Indexed: 04/10/2024] Open
Abstract
AIM Alpha-1-acid glycoprotein (AGP) is a highly glycosylated protein in human plasma and one of the most abundant acute phase proteins in humans. Glycosylation plays a crucial role in its biological functions, and alterations in AGP N-glycome have been associated with various diseases and inflammatory conditions. However, large-scale studies of AGP N-glycosylation in the general population are lacking. METHODS Using recently developed high-throughput glycoproteomic workflow for site-specific AGP N-glycosylation analysis, 803 individuals from the Croatian island of Korcula were analyzed and their AGP N-glycome data associated with biochemical and physiological traits, as well as different environmental factors. RESULTS After regression analysis, we found that AGP N-glycosylation is strongly associated with sex, somewhat less with age, along with multiple biochemical and physiological traits (e.g. BMI, triglycerides, uric acid, glucose, smoking status, fibrinogen). CONCLUSION For the first time we have extensively explored the inter-individual variability of AGP N-glycome in a general human population, demonstrating its changes with sex, age, biochemical, and physiological status of individuals, providing the baseline for future population and clinical studies.
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Affiliation(s)
- Frano Vučković
- Genos Glycoscience Research Laboratory, Borongajska cesta 83h, 10000 Zagreb, Croatia
| | - Mislav Novokmet
- Genos Glycoscience Research Laboratory, Borongajska cesta 83h, 10000 Zagreb, Croatia
| | - Dinko Šoić
- Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia
| | - Jerko Štambuk
- Genos Glycoscience Research Laboratory, Borongajska cesta 83h, 10000 Zagreb, Croatia
| | - Ivana Kolčić
- Department of Public Health, University of Split School of Medicine, Šoltanska ulica 2A, 21000 Split, Croatia
| | - Ozren Polašek
- Department of Public Health, University of Split School of Medicine, Šoltanska ulica 2A, 21000 Split, Croatia
- Algebra University College, Gradišćanska ulica 24, 10000 Zagreb, Croatia
| | - Gordan Lauc
- Genos Glycoscience Research Laboratory, Borongajska cesta 83h, 10000 Zagreb, Croatia
- Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia
| | - Olga Gornik
- Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia
| | - Toma Keser
- Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia
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8
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Anwar MA, Keshteli AH, Yang H, Wang W, Li X, Messier HM, Cullis PR, Borchers CH, Fraser R, Wishart DS. Blood-Based Multiomics-Guided Detection of a Precancerous Pancreatic Tumor. OMICS : A JOURNAL OF INTEGRATIVE BIOLOGY 2024; 28:182-192. [PMID: 38634790 DOI: 10.1089/omi.2023.0278] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/19/2024]
Abstract
Over a decade ago, longitudinal multiomics analysis was pioneered for early disease detection and individually tailored precision health interventions. However, high sample processing costs, expansive multiomics measurements along with complex data analysis have made this approach to precision/personalized medicine impractical. Here we describe in a case report, a more practical approach that uses fewer measurements, annual sampling, and faster decision making. We also show how this approach offers promise to detect an exceedingly rare and potentially fatal condition before it fully manifests. Specifically, we describe in the present case report how longitudinal multiomics monitoring (LMOM) helped detect a precancerous pancreatic tumor and led to a successful surgical intervention. The patient, enrolled in an annual blood-based LMOM since 2018, had dramatic changes in the June 2021 and 2022 annual metabolomics and proteomics results that prompted further clinical diagnostic testing for pancreatic cancer. Using abdominal magnetic resonance imaging, a 2.6 cm lesion in the tail of the patient's pancreas was detected. The tumor fluid from an aspiration biopsy had 10,000 times that of normal carcinoembryonic antigen levels. After the tumor was surgically resected, histopathological findings confirmed it was a precancerous pancreatic tumor. Postoperative omics testing indicated that most metabolite and protein levels returned to patient's 2018 levels. This case report illustrates the potentials of blood LMOM for precision/personalized medicine, and new ways of thinking medical innovation for a potentially life-saving early diagnosis of pancreatic cancer. Blood LMOM warrants future programmatic translational research with the goals of precision medicine, and individually tailored cancer diagnoses and treatments.
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Affiliation(s)
| | | | - Haiyan Yang
- Molecular You Corporation, Vancouver, British Columbia, Canada
| | - Windy Wang
- Molecular You Corporation, Vancouver, British Columbia, Canada
| | - Xukun Li
- Molecular You Corporation, Vancouver, British Columbia, Canada
| | - Helen M Messier
- Molecular You Corporation, Vancouver, British Columbia, Canada
- Fountain Life, Naples, Florida, USA
| | - Pieter R Cullis
- Molecular You Corporation, Vancouver, British Columbia, Canada
- Life Sciences Centre, University of British Columbia, Vancouver, British Columbia, Canada
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Christoph H Borchers
- Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, Montreal, Quebec, Canada
- Segal Cancer Proteomics Centre, Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, Quebec, Canada
- Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada
- Department of Pathology, McGill University, Montreal, Quebec, Canada
| | - Robert Fraser
- Molecular You Corporation, Vancouver, British Columbia, Canada
| | - David S Wishart
- Molecular You Corporation, Vancouver, British Columbia, Canada
- Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada
- Department of Computing Science, University of Alberta, Edmonton, Alberta, Canada
- Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada
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9
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He K, Baniasad M, Kwon H, Caval T, Xu G, Lebrilla C, Hommes DW, Bertozzi C. Decoding the glycoproteome: a new frontier for biomarker discovery in cancer. J Hematol Oncol 2024; 17:12. [PMID: 38515194 PMCID: PMC10958865 DOI: 10.1186/s13045-024-01532-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2023] [Accepted: 03/04/2024] [Indexed: 03/23/2024] Open
Abstract
Cancer early detection and treatment response prediction continue to pose significant challenges. Cancer liquid biopsies focusing on detecting circulating tumor cells (CTCs) and DNA (ctDNA) have shown enormous potential due to their non-invasive nature and the implications in precision cancer management. Recently, liquid biopsy has been further expanded to profile glycoproteins, which are the products of post-translational modifications of proteins and play key roles in both normal and pathological processes, including cancers. The advancements in chemical and mass spectrometry-based technologies and artificial intelligence-based platforms have enabled extensive studies of cancer and organ-specific changes in glycans and glycoproteins through glycomics and glycoproteomics. Glycoproteomic analysis has emerged as a promising tool for biomarker discovery and development in early detection of cancers and prediction of treatment efficacy including response to immunotherapies. These biomarkers could play a crucial role in aiding in early intervention and personalized therapy decisions. In this review, we summarize the significant advance in cancer glycoproteomic biomarker studies and the promise and challenges in integration into clinical practice to improve cancer patient care.
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Affiliation(s)
- Kai He
- James Comprehensive Cancer Center, The Ohio State University, Columbus, USA.
| | | | - Hyunwoo Kwon
- James Comprehensive Cancer Center, The Ohio State University, Columbus, USA
| | | | - Gege Xu
- InterVenn Biosciences, South San Francisco, USA
| | - Carlito Lebrilla
- Department of Biochemistry and Molecular Medicine, UC Davis Health, Sacramento, USA
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10
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Liu L, Liu L, Wang Y, Fang Z, Bian Y, Zhang W, Wang Z, Gao X, Zhao C, Tian M, Liu X, Qin H, Guo Z, Liang X, Dong M, Nie Y, Ye M. Robust Glycoproteomics Platform Reveals a Tetra-Antennary Site-Specific Glycan Capping with Sialyl-Lewis Antigen for Early Detection of Gastric Cancer. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2306955. [PMID: 38084450 PMCID: PMC10916543 DOI: 10.1002/advs.202306955] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/21/2023] [Revised: 11/16/2023] [Indexed: 03/07/2024]
Abstract
The lack of efficient biomarkers for the early detection of gastric cancer (GC) contributes to its high mortality rate, so it is crucial to discover novel diagnostic targets for GC. Recent studies have implicated the potential of site-specific glycans in cancer diagnosis, yet it is challenging to perform highly reproducible and sensitive glycoproteomics analysis on large cohorts of samples. Here, a highly robust N-glycoproteomics (HRN) platform comprising an automated enrichment method, a stable microflow LC-MS/MS system, and a sensitive glycopeptide-spectra-deciphering tool is developed for large-scale quantitative N-glycoproteome analysis. The HRN platform is applied to analyze serum N-glycoproteomes of 278 subjects from three cohorts to investigate glycosylation changes of GC. It identifies over 20 000 unique site-specific glycans from discovery and validation cohorts, and determines four site-specific glycans as biomarker candidates. One candidate has branched tetra-antennary structure capping with sialyl-Lewis antigen, and it significantly outperforms serum CEA with AUC values > 0.89 compared against < 0.67 for diagnosing early-stage GC. The four-marker panel can provide improved diagnostic performances. Besides, discrimination powers of four candidates are also testified with a verification cohort using PRM strategy. This findings highlight the value of this strong tool in analyzing aberrant site-specific glycans for cancer detection.
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Affiliation(s)
- Luyao Liu
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China
- University of Chinese Academy of SciencesBeijing101408China
| | - Lei Liu
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China
- University of Chinese Academy of SciencesBeijing101408China
| | - Yan Wang
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China
| | - Zheng Fang
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China
| | - Yangyang Bian
- The College of Life SciencesNorthwest UniversityXi'an710127China
| | - Wenyao Zhang
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive DiseasesFourth Military Medical UniversityXi'an710068China
| | - Zhongyu Wang
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China
| | - Xianchun Gao
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive DiseasesFourth Military Medical UniversityXi'an710068China
| | - Changrui Zhao
- MOE Key Laboratory of Bio‐Intelligent Manufacturing, School of BioengineeringDalian University of TechnologyDalian116024China
| | - Miaomiao Tian
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive DiseasesFourth Military Medical UniversityXi'an710068China
| | - Xiaoyan Liu
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China
| | - Hongqiang Qin
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China
| | - Zhimou Guo
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China
| | - Xinmiao Liang
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China
| | - Mingming Dong
- MOE Key Laboratory of Bio‐Intelligent Manufacturing, School of BioengineeringDalian University of TechnologyDalian116024China
| | - Yongzhan Nie
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive DiseasesFourth Military Medical UniversityXi'an710068China
| | - Mingliang Ye
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical PhysicsChinese Academy of SciencesDalian116023China
- University of Chinese Academy of SciencesBeijing101408China
- State Key Laboratory of Medical ProteomicsBeijing102206China
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11
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Šeba T, Kerep R, Weitner T, Šoić D, Keser T, Lauc G, Gabričević M. Influence of Desialylation on the Drug Binding Affinity of Human Alpha-1-Acid Glycoprotein Assessed by Microscale Thermophoresis. Pharmaceutics 2024; 16:230. [PMID: 38399284 PMCID: PMC10893521 DOI: 10.3390/pharmaceutics16020230] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2023] [Revised: 01/22/2024] [Accepted: 01/31/2024] [Indexed: 02/25/2024] Open
Abstract
Human serum alpha-1-acid glycoprotein (AAG) is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. The sialic acid groups that terminate the N-glycan chains of AAG have been reported to change in response to numerous health conditions and may have an impact on the binding of drugs to AAG. In this study, we quantified the binding between native and desialylated AAG and seven drugs from different pharmacotherapeutic groups (carvedilol, diltiazem, dipyridamole, imipramine, lidocaine, propranolol, vinblastine) using microscale thermophoresis (MST). This method was chosen due to its robustness and high sensitivity, allowing precise quantification of molecular interactions based on the thermophoretic movement of fluorescent molecules. Detailed glycan analysis of native and desialylated AAG showed over 98% reduction in sialic acid content for the enzymatically desialylated AAG. The MST results indicate that desialylation generally alters the binding affinity between AAG and drugs, leading to either an increase or decrease in Kd values, probably due to conformational changes of AAG caused by the different sialic acid content. This effect is also reflected in an increased denaturation temperature of desialylated AAG. Our findings indicate that desialylation impacts free drug concentrations differently, depending on the binding affinity of the drug with AAG relative to human serum albumin (HSA). For drugs such as dipyridamole, lidocaine, and carvedilol, which have a higher affinity for AAG, desialylation significantly changes free drug concentrations. In contrast, drugs such as propranolol, imipramine, and vinblastine, which have a strong albumin binding, show only minimal changes. It is noteworthy that the free drug concentration of dipyridamole is particularly sensitive to changes in AAG concentration and glycosylation, with a decrease of up to 15% being observed, underscoring the need for dosage adjustments in personalized medicine.
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Affiliation(s)
- Tino Šeba
- Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, 10000 Zagreb, Croatia; (T.Š.); (R.K.); (T.W.)
| | - Robert Kerep
- Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, 10000 Zagreb, Croatia; (T.Š.); (R.K.); (T.W.)
| | - Tin Weitner
- Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, 10000 Zagreb, Croatia; (T.Š.); (R.K.); (T.W.)
| | - Dinko Šoić
- Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, 10000 Zagreb, Croatia; (D.Š.); (T.K.); (G.L.)
| | - Toma Keser
- Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, 10000 Zagreb, Croatia; (D.Š.); (T.K.); (G.L.)
| | - Gordan Lauc
- Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, 10000 Zagreb, Croatia; (D.Š.); (T.K.); (G.L.)
| | - Mario Gabričević
- Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovačića 1, 10000 Zagreb, Croatia; (T.Š.); (R.K.); (T.W.)
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12
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Zhu Y. Plasma/Serum Proteomics based on Mass Spectrometry. Protein Pept Lett 2024; 31:192-208. [PMID: 38869039 PMCID: PMC11165715 DOI: 10.2174/0109298665286952240212053723] [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: 11/22/2023] [Revised: 01/22/2024] [Accepted: 01/31/2024] [Indexed: 06/14/2024]
Abstract
Human blood is a window of physiology and disease. Examination of biomarkers in blood is a common clinical procedure, which can be informative in diagnosis and prognosis of diseases, and in evaluating treatment effectiveness. There is still a huge demand on new blood biomarkers and assays for precision medicine nowadays, therefore plasma/serum proteomics has attracted increasing attention in recent years. How to effectively proceed with the biomarker discovery and clinical diagnostic assay development is a question raised to researchers who are interested in this area. In this review, we comprehensively introduce the background and advancement of technologies for blood proteomics, with a focus on mass spectrometry (MS). Analyzing existing blood biomarkers and newly-built diagnostic assays based on MS can shed light on developing new biomarkers and analytical methods. We summarize various protein analytes in plasma/serum which include total proteome, protein post-translational modifications, and extracellular vesicles, focusing on their corresponding sample preparation methods for MS analysis. We propose screening multiple protein analytes in the same set of blood samples in order to increase success rate for biomarker discovery. We also review the trends of MS techniques for blood tests including sample preparation automation, and further provide our perspectives on their future directions.
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Affiliation(s)
- Yiying Zhu
- Department of Chemistry, Tsinghua University, Beijing, China
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13
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Lin T, Chen Z, Luo M, Zhao Y, Zeng W, Zheng S, Su T, Zhong Y, Wang S, Jin Y, Hu L, Zhao W, Li J, Wang X, Wu C, Li D, Liu F, Li G, Yang H, Zhang Y. Characterization of site-specific N-glycosylation signatures of isolated uromodulin from human urine. Analyst 2023; 148:5041-5049. [PMID: 37667671 DOI: 10.1039/d3an01018j] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/06/2023]
Abstract
Uromodulin (Umod, Tamm-Horsfall protein) is the most abundant urinary N-glycoprotein produced exclusively by the kidney. It can form filaments to antagonize the adhesion of uropathogens. However, the site-specific N-glycosylation signatures of Umod in healthy individuals and patients with IgA nephropathy (IgAN) remain poorly understood due to the lack of suitable isolation and analytical methods. In this study, we first presented a simple and fast method based on diatomaceous earth adsorption to isolate Umod. These isolated glycoproteins were digested by trypsin and/or Glu-C. Intact N-glycopeptides with or without HILIC enrichment were analyzed using our developed EThcD-sceHCD-MS/MS. Based on the optimized workflow, we identified a total of 780 unique intact N-glycopeptides (7 N-glycosites and 152 N-glycan compositions) from healthy individuals. As anticipated, these glycosites exhibited glycoform heterogeneity. Almost all N-glycosites were modified completely by the complex type, except for one N-glycosite (N275), which was nearly entirely occupied by the high-mannose type for mediating Umod's antiadhesive activity. Then, we compared the N-glycosylation of Umod between healthy controls (n = 9) and IgAN patients (n = 9). The N-glycosylation of Umod in IgAN patients will drastically decrease and be lost. Finally, we profiled the most comprehensive site-specific N-glycosylation map of Umod and revealed its alterations in IgAN patients. Our method provides a high-throughput workflow for characterizing the N-glycosylation of Umod, which can aid in understanding its roles in physiology and pathology, as well as serving as a potential diagnostic tool for evolution of renal tubular function.
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Affiliation(s)
- Tianhai Lin
- Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
- Department of Urology, West China Hospital, Sichuan University, Chengdu 610041, China
| | - Zhuo Chen
- Transplant Center and NHC Key Lab of Transplant Engineering and Immunology, Regenerative Medical Research Center, West China Hospital, Sichuan University, Chengdu 610041, China
| | - Mengqi Luo
- Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
| | - Yang Zhao
- Technology Innovation Center of Mass Spectrometry for State Market Regulation, Center for Advanced Measurement Science, National Institute of Metrology, Beijing 100029, China
| | - Wenjuan Zeng
- Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
| | - Shanshan Zheng
- Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
| | - Tao Su
- Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
| | - Yi Zhong
- Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
| | - Shisheng Wang
- Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
| | - Youmei Jin
- Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
| | - Liqiang Hu
- Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
| | - Wanjun Zhao
- Division of Thyroid Surgery, Department of General Surgery of Nursing, West China Hospital, Sichuan University, Chengdu 610041, China
| | - Jiaxu Li
- School of Nursing, Chengde Medical University, Chengde, Hebei 067000, China
| | - Xuanyi Wang
- Mingde College, Zhangjiakou University, Zhangjiakou, Hebei 075000, China
| | - Changwei Wu
- Renal Department and Institute of Nephrology, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Sichuan Clinical Research Center for Kidney Diseases, Chengdu 611731, China.
| | - Dapeng Li
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
| | - Fang Liu
- Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
| | - Guisen Li
- Renal Department and Institute of Nephrology, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Sichuan Clinical Research Center for Kidney Diseases, Chengdu 611731, China.
| | - Hao Yang
- Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
- Transplant Center and NHC Key Lab of Transplant Engineering and Immunology, Regenerative Medical Research Center, West China Hospital, Sichuan University, Chengdu 610041, China
| | - Yong Zhang
- Department of Nephrology and Institutes for Systems Genetics, Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
- Transplant Center and NHC Key Lab of Transplant Engineering and Immunology, Regenerative Medical Research Center, West China Hospital, Sichuan University, Chengdu 610041, China
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14
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Zhang Y, Minami R, Tatsuno R, Gao W, Ueno M, Yamada A, Yoshida A, Sedanza MG, Arima K, Takatani T, Yamaguchi K, Oshima Y, Arakawa O. Wheat germ agglutinin affinity chromatography enrichment and glyco-proteomic characterization of tetrodotoxin-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes). Biosci Biotechnol Biochem 2023; 87:1155-1168. [PMID: 37458754 DOI: 10.1093/bbb/zbad095] [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: 05/03/2023] [Accepted: 07/07/2023] [Indexed: 09/24/2023]
Abstract
Efficient enrichment of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes) was achieved by ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography. The enrichment efficiency was validated by ultrafiltration-LC/MS-based TTX-binding assay and proteomics. Major proteins in the WGA-bound fraction were identified as isoform X1 (125 kDa) and X2 variants (88 and 79 kDa) derived from pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) 1-like gene (LOC101075943). The 125-kDa X1 protein was found to be a novel member of the lipocalin family, having three tandemly repeated domains. X2 variants, X2α and X2β, were estimated to have two domains, and X2β is structurally related to Takifugu pardalis PSTBP2 in their domain type and arrangement. Among 11 potential N-glycosylation sites in the X2 precursor, 5 N-glycosylated Asn residues (N55, N89, N244, N308, and N449) were empirically determined. Structural relationships among PSTBP homologs and complexity of their proteoforms are discussed.
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Affiliation(s)
- Yafei Zhang
- Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Bunkyo-machi, Nagasaki, Japan
| | - Ryoma Minami
- Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Bunkyo-machi, Nagasaki, Japan
- Division of Transcriptomics, Medical Institute of Bioregulation, Kyushu University, Maidashi, Higashi-ku, Fukuoka, Japan
| | - Ryohei Tatsuno
- National Fisheries University, Japan Fisheries Research and Education Agency, Nagatahonmachi, Shimonoseki, Yamaguchi, Japan
| | - Wei Gao
- Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Bunkyo-machi, Nagasaki, Japan
- Dalian Blue Peptide Technology Research & Development Co., Ltd, Dalian, China
| | - Mikinori Ueno
- Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Bunkyo-machi, Nagasaki, Japan
| | - Akinori Yamada
- Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Bunkyo-machi, Nagasaki, Japan
| | - Asami Yoshida
- Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Bunkyo-machi, Nagasaki, Japan
| | - Mary Grace Sedanza
- Institute of Aquaculture, College of Fisheries and Ocean Sciences, University of the Philippines Visayas, Miagao, Iloilo, Philippines
| | - Kazunari Arima
- Department of Chemistry, Graduate School of Science and Engineering, Kagoshima University, Korimoto, Kagoshima, Japan
| | - Tomohiro Takatani
- Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Bunkyo-machi, Nagasaki, Japan
| | - Kenichi Yamaguchi
- Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Bunkyo-machi, Nagasaki, Japan
| | - Yuji Oshima
- Laboratory of Marine Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka, Japan
| | - Osamu Arakawa
- Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Bunkyo-machi, Nagasaki, Japan
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15
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Suttapitugsakul S, Matsumoto Y, Aryal RP, Cummings RD. Large-Scale and Site-Specific Mapping of the Murine Brain O-Glycoproteome with IMPa. Anal Chem 2023; 95:13423-13430. [PMID: 37624755 PMCID: PMC10501376 DOI: 10.1021/acs.analchem.3c00408] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2023] [Accepted: 07/16/2023] [Indexed: 08/27/2023]
Abstract
Altered protein glycosylation is typically associated with cognitive defects and other phenotypes, but there is a lack of knowledge about the brain glycoproteome. Here, we used the newly available O-glycoprotease IMPa from Pseudomonas aeruginosa for comprehensive O-glycoproteomic analyses of the mouse brain. In this approach, total tryptic glycopeptides were prepared, extracted, purified, and conjugated to a solid support before an enzymatic cleavage by IMPa. O-glycopeptides were analyzed by electron-transfer/higher-energy collision dissociation (EThcD), which permits site-specific and global analysis of all types of O-glycans. We developed two complementary approaches for the analysis of the total O-glycoproteome using HEK293 cells and derivatives. The results demonstrated that IMPa and EThcD facilitate the confident localization of O-glycans on glycopeptides. We then applied these approaches to characterize the O-glycoproteome of the mouse brain, which revealed the high frequency of various sialylated O-glycans along with the unusual presence of the Tn antigen. Unexpectedly, the results demonstrated that glycoproteins in the brain O-glycoproteome only partly overlap with those reported for the brain N-glycoproteome. These approaches will aid in identifying the novel O-glycoproteomes of different cells and tissues and foster clinical and translational insights into the functions of protein O-glycosylation in the brain and other organs.
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Affiliation(s)
- Suttipong Suttapitugsakul
- Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, Massachusetts 02215, United States
| | | | - Rajindra P. Aryal
- Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, Massachusetts 02215, United States
| | - Richard D. Cummings
- Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, Massachusetts 02215, United States
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16
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Abbas M, Alossaimi MA, Altamimi ASA, Alajaji M, Watson DG, Shah SI, Shah Y, Anwar MS. Determination of α1-acid glycoprotein (AGP) concentration by HPLC in patients following local infiltration analgesia for primary total hip arthroplasty and its relation to ropivacaine (total and unbound). Front Pharmacol 2023; 14:1145962. [PMID: 37456752 PMCID: PMC10345198 DOI: 10.3389/fphar.2023.1145962] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2023] [Accepted: 05/16/2023] [Indexed: 07/18/2023] Open
Abstract
Introduction: This study was performed to determine the levels of α1-acid glycoprotein (AGP) in old-age patients undergoing total hip arthroplasty. AGP is considered an acute phase protein produced during the acute phase reaction in the body to various stimuli; their proper monitoring is thus important. Methods: In order to study how AGP concentrations in old age patients change in response to surgical stress (total hip arthroplasty), a high-performance liquid chromatography assay was performed to measure AGP levels. AGP was isolated from the plasma by adding perchloric acid and was analyzed using PLRP-S 4000°A column. The mobile phase consisted of 1 mL TFA/L of water (Solvent A pH 2) and 1 mL TFA/L of acetonitrile (Solvent B). The gradient used was as follows: 0 min 18% B and 82% A, 15 min 60% B and 40% A, and 17 min 60% B and 40% A followed by column re-equilibration for 7 min before the next injection. AGP peak was obtained between 8.8 and 8.9 min. The method was fully optimised according to established guidelines. Results: The data obtained were analyzed on ChromQuest software. AGP concentrations were determined in all samples, including baseline and samples taken at different timed intervals. The peak for AGP was obtained between 8.8 and 8.9 min for both standard AGP and patient plasma. The graphs indicate that AGP concentration in almost all patient samples increased considerably, especially after 4 h and 24 h-for example, initial concentration in patient 1 was 10.36 mg/100 mL but, after 24 h, increased to 23.50 mg/100 mL. There was thus almost a 13 mg/100 mL increase in 24 h, which is confirmed by AGP concentration increasing after various conditions, including surgery. The increased plasma protein binding was comparatively associated with the unchanged free fraction of the drug. Conclusion: This surgically induced increase in AGP concentration resulted in increased plasma protein binding of the drug (ropivacaine), which in turn kept the free portion of ropivacaine stable during the postoperative period.
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Affiliation(s)
- Muhammad Abbas
- Department of Pharmacy, Abdul Wali Khan University Mardan, Mardan, Pakistan
| | - Manal A. Alossaimi
- Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdul Aziz University, Al-Kharj, Saudi Arabia
| | - Abdulmalik S. A. Altamimi
- Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdul Aziz University, Al-Kharj, Saudi Arabia
| | - Mai Alajaji
- College of Pharmacy, King Abdullah International Medical Research Center, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia
| | - David G. Watson
- Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom
| | - Sayyed I. Shah
- Department of Pharmacy, Abdul Wali Khan University Mardan, Mardan, Pakistan
| | - Yasar Shah
- Department of Pharmacy, Abdul Wali Khan University Mardan, Mardan, Pakistan
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17
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Kerep R, Šeba T, Borko V, Weitner T, Keser T, Lauc G, Gabričević M. Potential Clinically Relevant Effects of Sialylation on Human Serum AAG-Drug Interactions Assessed by Isothermal Titration Calorimetry: Insight into Pharmacoglycomics? Int J Mol Sci 2023; 24:8472. [PMID: 37239819 PMCID: PMC10218007 DOI: 10.3390/ijms24108472] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2023] [Revised: 04/18/2023] [Accepted: 04/25/2023] [Indexed: 05/28/2023] Open
Abstract
Human serum alpha-1 acid glycoprotein is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. It has been reported that the sialic acid groups that terminate the N-glycan chains of alpha-1 acid glycoprotein change in response to certain health conditions and may have a major impact on drug binding to alpha-1 acid glycoprotein. The interaction between native or desialylated alpha-1 acid glycoprotein and four representative drugs-clindamycin, diltiazem, lidocaine, and warfarin-was quantitatively evaluated using isothermal titration calorimetry. The calorimetry assay used here is a convenient and widely used approach to directly measure the amount of heat released or absorbed during the association processes of biomolecules in solution and to quantitatively estimate the thermodynamics of the interaction. The results showed that the binding of drugs with alpha-1 acid glycoprotein were enthalpy-driven exothermic interactions, and the binding affinity was in the range of 10-5-10-6 M. Desialylated alpha-1 acid glycoprotein showed significantly different binding with diltiazem, lidocaine, and warfarin compared with native alpha-1 acid glycoprotein, whereas clindamycin showed no significant difference. Therefore, a different degree of sialylation may result in different binding affinities, and the clinical significance of changes in sialylation or glycosylation of alpha-1 acid glycoprotein in general should not be neglected.
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Affiliation(s)
- Robert Kerep
- Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Croatia
| | - Tino Šeba
- Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Croatia
| | - Valentina Borko
- Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Croatia
| | - Tin Weitner
- Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Croatia
| | - Toma Keser
- Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Croatia
| | - Gordan Lauc
- Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Croatia
| | - Mario Gabričević
- Department of General and Inorganic Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000 Zagreb, Croatia
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18
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van der Burgt Y, Wuhrer M. The role of clinical glyco(proteo)mics in precision medicine. Mol Cell Proteomics 2023:100565. [PMID: 37169080 DOI: 10.1016/j.mcpro.2023.100565] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Revised: 04/12/2023] [Accepted: 05/02/2023] [Indexed: 05/13/2023] Open
Abstract
Glycoproteomics reveals site-specific O- and N-glycosylation that may influence protein properties including binding, activity and half-life. The increasingly mature toolbox with glycomic- and glycoproteomic strategies is applied for the development of biopharmaceuticals and discovery and clinical evaluation of glycobiomarkers in various disease fields. Notwithstanding the contributions of glycoscience in identifying new drug targets, the current report is focused on the biomarker modality that is of interest for diagnostic and monitoring purposes. To this end it is noted that the identification of biomarkers has received more attention than corresponding quantification. Most analytical methods are very efficient in detecting large numbers of analytes but developments to accurately quantify these have so far been limited. In this perspective a parallel is made with earlier proposed tiers for protein quantification using mass spectrometry. Moreover, the foreseen reporting of multimarker readouts is discussed to describe an individual's health or disease state and their role in clinical decision-making. The potential of longitudinal sampling and monitoring of glycomic features for diagnosis and treatment monitoring is emphasized. Finally, different strategies that address quantification of a multimarker panel will be discussed.
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Affiliation(s)
- Yuri van der Burgt
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands.
| | - Manfred Wuhrer
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands
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19
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Hu M, Zhang R, Yang J, Zhao C, Liu W, Huang Y, Lyu H, Xiao S, Guo D, Zhou C, Tang J. The role of N-glycosylation modification in the pathogenesis of liver cancer. Cell Death Dis 2023; 14:222. [PMID: 36990999 PMCID: PMC10060418 DOI: 10.1038/s41419-023-05733-z] [Citation(s) in RCA: 17] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2022] [Revised: 03/02/2023] [Accepted: 03/13/2023] [Indexed: 03/31/2023]
Abstract
N-glycosylation is one of the most common types of protein modifications and it plays a vital role in normal physiological processes. However, aberrant N-glycan modifications are closely associated with the pathogenesis of diverse diseases, including processes such as malignant transformation and tumor progression. It is known that the N-glycan conformation of the associated glycoproteins is altered during different stages of hepatocarcinogenesis. Characterizing the heterogeneity and biological functions of glycans in liver cancer patients will facilitate a deeper understanding of the molecular mechanisms of liver injury and hepatocarcinogenesis. In this article, we review the role of N-glycosylation in hepatocarcinogenesis, focusing on epithelial-mesenchymal transition, extracellular matrix changes, and tumor microenvironment formation. We highlight the role of N-glycosylation in the pathogenesis of liver cancer and its potential applications in the treatment or diagnosis of liver cancer.
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Affiliation(s)
- Mengyu Hu
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Rui Zhang
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Jiaren Yang
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Chenshu Zhao
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Wei Liu
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Yuan Huang
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Hao Lyu
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Shuai Xiao
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Dong Guo
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Cefan Zhou
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China.
| | - Jingfeng Tang
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China.
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20
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Hu M, Zhang R, Yang J, Zhao C, Liu W, Huang Y, Lyu H, Xiao S, Guo D, Zhou C, Tang J. The role of N-glycosylation modification in the pathogenesis of liver cancer. Cell Death Dis 2023; 14:222. [PMID: 36990999 DOI: 10.1038/s41419-023-05733-z.pmid:] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2022] [Revised: 03/02/2023] [Accepted: 03/13/2023] [Indexed: 10/14/2024]
Abstract
N-glycosylation is one of the most common types of protein modifications and it plays a vital role in normal physiological processes. However, aberrant N-glycan modifications are closely associated with the pathogenesis of diverse diseases, including processes such as malignant transformation and tumor progression. It is known that the N-glycan conformation of the associated glycoproteins is altered during different stages of hepatocarcinogenesis. Characterizing the heterogeneity and biological functions of glycans in liver cancer patients will facilitate a deeper understanding of the molecular mechanisms of liver injury and hepatocarcinogenesis. In this article, we review the role of N-glycosylation in hepatocarcinogenesis, focusing on epithelial-mesenchymal transition, extracellular matrix changes, and tumor microenvironment formation. We highlight the role of N-glycosylation in the pathogenesis of liver cancer and its potential applications in the treatment or diagnosis of liver cancer.
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Affiliation(s)
- Mengyu Hu
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Rui Zhang
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Jiaren Yang
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Chenshu Zhao
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Wei Liu
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Yuan Huang
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Hao Lyu
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Shuai Xiao
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Dong Guo
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China
| | - Cefan Zhou
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China.
| | - Jingfeng Tang
- National "111" Center for Cellular Regulation and Molecular Pharmaceutics, Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Wuhan, China.
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21
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Trbojević-Akmačić I, Vučković F, Pribić T, Vilaj M, Černigoj U, Vidič J, Šimunović J, Kępka A, Kolčić I, Klarić L, Novokmet M, Pučić-Baković M, Rapp E, Štrancar A, Polašek O, Wilson JF, Lauc G. Comparative analysis of transferrin and IgG N-glycosylation in two human populations. Commun Biol 2023; 6:312. [PMID: 36959410 PMCID: PMC10036557 DOI: 10.1038/s42003-023-04685-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2022] [Accepted: 03/09/2023] [Indexed: 03/25/2023] Open
Abstract
Human plasma transferrin (Tf) N-glycosylation has been mostly studied as a marker for congenital disorders of glycosylation, alcohol abuse, and hepatocellular carcinoma. However, inter-individual variability of Tf N-glycosylation is not known, mainly due to technical limitations of Tf isolation in large-scale studies. Here, we present a highly specific robust high-throughput approach for Tf purification from human blood plasma and detailed characterization of Tf N-glycosylation on the level of released glycans by ultra-high-performance liquid chromatography based on hydrophilic interactions and fluorescence detection (HILIC-UHPLC-FLD), exoglycosidase sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We perform a large-scale comparative study of Tf and immunoglobulin G (IgG) N-glycosylation analysis in two human populations and demonstrate that Tf N-glycosylation is associated with age and sex, along with multiple biochemical and physiological traits. Observed association patterns differ compared to the IgG N-glycome corroborating tissue-specific N-glycosylation and specific N-glycans' role in their distinct physiological functions.
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Affiliation(s)
| | | | - Tea Pribić
- Genos Glycoscience Research Laboratory, Zagreb, Croatia
| | - Marija Vilaj
- Genos Glycoscience Research Laboratory, Zagreb, Croatia
| | - Urh Černigoj
- BIA Separations d.o.o., a Sartorius company, Ajdovščina, Slovenia
| | - Jana Vidič
- BIA Separations d.o.o., a Sartorius company, Ajdovščina, Slovenia
| | | | - Agnieszka Kępka
- Genos Glycoscience Research Laboratory, Zagreb, Croatia
- Department of Immunology, Faculty of Biology, Institute of Zoology, University of Warsaw, Warsaw, Poland
| | - Ivana Kolčić
- Department of Public Health, University of Split School of Medicine, Split, Croatia
- Algebra University College, Zagreb, Croatia
| | - Lucija Klarić
- MRC Human Genetics Unit, Institute for Genetics and Cancer, University of Edinburgh, Edinburgh, UK
| | | | | | - Erdmann Rapp
- Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany
- glyXera GmbH, Magdeburg, Germany
| | - Aleš Štrancar
- BIA Separations d.o.o., a Sartorius company, Ajdovščina, Slovenia
| | - Ozren Polašek
- Department of Public Health, University of Split School of Medicine, Split, Croatia
- Algebra University College, Zagreb, Croatia
| | - James F Wilson
- MRC Human Genetics Unit, Institute for Genetics and Cancer, University of Edinburgh, Edinburgh, UK
- Centre for Global Health Research, Usher Institute, University of Edinburgh, Edinburgh, UK
| | - Gordan Lauc
- Genos Glycoscience Research Laboratory, Zagreb, Croatia.
- Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia.
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22
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Wong TL, Mooney BP, Cavallero GJ, Guan M, Li L, Zaia J, Wan XF. Glycoproteomic Analyses of Influenza A Viruses Using timsTOF Pro MS. J Proteome Res 2023; 22:62-77. [PMID: 36480915 DOI: 10.1021/acs.jproteome.2c00469] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
N-Linked glycosylation in hemagglutinin and neuraminidase glycoproteins of influenza viruses affects antigenic and receptor binding properties, and precise analyses of site-specific glycoforms in these proteins are critical in understanding the antigenic and immunogenic properties of influenza viruses. In this study, we developed a glycoproteomic approach by using a timsTOF Pro mass spectrometer (MS) to determine the abundance and heterogeneity of site-specific glycosylation for influenza glycoproteins. Compared with a Q Exactive HF MS, the timsTOF Pro MS method without the hydrophilic interaction liquid chromatography column enrichment achieved similar glycopeptide coverage and quantities but was more effective in identifying low-abundance glycopeptides. We quantified the distributions of intact site-specific glycopeptides in hemagglutinin of A/chicken/Wuxi/0405005/2013 (H7N9) and A/mute swan/Rhode Island/A00325125/2008 (H7N3). Results showed that hemagglutinin for both viruses had complex N-glycans at N22, N38, N240, and N483 but only high-mannose glycans at N411 and, however, that the type and quantities of glycans were distinct between these viruses. Collisional cross section (CCS) provided by the ion mobility spectrometry from the timsTOF Pro MS data differentiated sialylation linkages of the glycopeptides. In summary, timsTOF Pro MS method can quantify intact site-specific glycans for influenza glycoproteins without enrichment and thus facilitate influenza vaccine development and production.
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Affiliation(s)
- Tin Long Wong
- Center for Influenza and Emerging Infectious Diseases, University of Missouri, Columbia, Missouri65211, United States.,Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, Missouri65211, United States.,Bond Life Sciences Center, University of Missouri, Columbia, Missouri65211, United States
| | - Brian P Mooney
- Department of Biochemistry and Charles W. Gehrke Proteomics Center, University of Missouri, Columbia, Missouri65211, United States
| | - Gustavo J Cavallero
- Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, Massachusetts02118, United States
| | - Minhui Guan
- Center for Influenza and Emerging Infectious Diseases, University of Missouri, Columbia, Missouri65211, United States.,Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, Missouri65211, United States.,Bond Life Sciences Center, University of Missouri, Columbia, Missouri65211, United States
| | - Lei Li
- Department of Chemistry, Georgia State University, Atlanta, Georgia30302, United States
| | - Joseph Zaia
- Department of Biochemistry, Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, Massachusetts02118, United States
| | - Xiu-Feng Wan
- Center for Influenza and Emerging Infectious Diseases, University of Missouri, Columbia, Missouri65211, United States.,Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, Missouri65211, United States.,Bond Life Sciences Center, University of Missouri, Columbia, Missouri65211, United States.,Department of Electrical Engineering & Computer Science, College of Engineering, University of Missouri, Columbia, Missouri65211, United States
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23
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Susceptibility of Human Plasma N-glycome to Low-Calorie and Different Weight-Maintenance Diets. Int J Mol Sci 2022; 23:ijms232415772. [PMID: 36555411 PMCID: PMC9779867 DOI: 10.3390/ijms232415772] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2022] [Revised: 12/06/2022] [Accepted: 12/08/2022] [Indexed: 12/15/2022] Open
Abstract
Aberrant plasma protein glycosylation is associated with a wide range of diseases, including diabetes, cardiovascular, and immunological disorders. To investigate plasma protein glycosylation alterations due to weight loss and successive weight-maintenance diets, 1850 glycomes from participants of the Diogenes study were analyzed using Ultra-High-Performance Liquid Chromatography (UHPLC). The Diogenes study is a large dietary intervention study in which participants were subjected to a low-calorie diet (LCD) followed by one of five different weight-maintenance diets in a period of 6 months. The most notable alterations of the plasma glycome were 8 weeks after the subjects engaged in the LCD; a significant increase in low-branched glycan structures, accompanied by a decrease in high-branched glycan structures. After the LCD period, there was also a significant rise in N-glycan structures with antennary fucose. Interestingly, we did not observe significant changes between different diets, and almost all effects we observed immediately after the LCD period were annulled during the weight-maintenance diets period.
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24
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Trbojević-Akmačić I, Lageveen-Kammeijer GSM, Heijs B, Petrović T, Deriš H, Wuhrer M, Lauc G. High-Throughput Glycomic Methods. Chem Rev 2022; 122:15865-15913. [PMID: 35797639 PMCID: PMC9614987 DOI: 10.1021/acs.chemrev.1c01031] [Citation(s) in RCA: 37] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Glycomics aims to identify the structure and function of the glycome, the complete set of oligosaccharides (glycans), produced in a given cell or organism, as well as to identify genes and other factors that govern glycosylation. This challenging endeavor requires highly robust, sensitive, and potentially automatable analytical technologies for the analysis of hundreds or thousands of glycomes in a timely manner (termed high-throughput glycomics). This review provides a historic overview as well as highlights recent developments and challenges of glycomic profiling by the most prominent high-throughput glycomic approaches, with N-glycosylation analysis as the focal point. It describes the current state-of-the-art regarding levels of characterization and most widely used technologies, selected applications of high-throughput glycomics in deciphering glycosylation process in healthy and disease states, as well as future perspectives.
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Affiliation(s)
| | | | - Bram Heijs
- Center
for Proteomics and Metabolomics, Leiden
University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
| | - Tea Petrović
- Genos,
Glycoscience Research Laboratory, Borongajska cesta 83H, 10 000 Zagreb, Croatia
| | - Helena Deriš
- Genos,
Glycoscience Research Laboratory, Borongajska cesta 83H, 10 000 Zagreb, Croatia
| | - Manfred Wuhrer
- Center
for Proteomics and Metabolomics, Leiden
University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
| | - Gordan Lauc
- Genos,
Glycoscience Research Laboratory, Borongajska cesta 83H, 10 000 Zagreb, Croatia
- Faculty
of Pharmacy and Biochemistry, University
of Zagreb, A. Kovačića 1, 10 000 Zagreb, Croatia
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25
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Petralia LMC, Santha E, Behrens AJ, Nguyen DL, Ganatra MB, Taron CH, Khatri V, Kalyanasundaram R, van Diepen A, Hokke CH, Foster JM. Alteration of rhesus macaque serum N-glycome during infection with the human parasitic filarial nematode Brugia malayi. Sci Rep 2022; 12:15763. [PMID: 36131114 PMCID: PMC9491660 DOI: 10.1038/s41598-022-19964-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2022] [Accepted: 09/07/2022] [Indexed: 11/09/2022] Open
Abstract
Serum N-glycan profiling studies during the past decades have shown robust associations between N-glycan changes and various biological conditions, including infections, in humans. Similar studies are scarcer for other mammals, despite the tremendous potential of serum N-glycans as biomarkers for infectious diseases in animal models of human disease and in the veterinary context. To expand the knowledge of serum N-glycan profiles in important mammalian model systems, in this study, we combined MALDI-TOF-MS analysis and HILIC-UPLC profiling of released N-glycans together with glycosidase treatments to characterize the glycan structures present in rhesus macaque serum. We used this baseline to monitor changes in serum N-glycans during infection with Brugia malayi, a parasitic nematode of humans responsible for lymphatic filariasis, in a longitudinal cohort of infected rhesus macaques. Alterations of the HILIC-UPLC profile, notably of abundant structures, became evident as early as 5 weeks post-infection. Given its prominent role in the immune response, contribution of immunoglobulin G to serum N-glycans was investigated. Finally, comparison with similar N-glycan profiling performed during infection with the dog heartworm Dirofilaria immitis suggests that many changes observed in rhesus macaque serum N-glycans are specific for lymphatic filariasis.
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Affiliation(s)
- Laudine M C Petralia
- Division of Protein Expression and Modification, New England Biolabs, Ipswich, MA, 01938, USA.
- Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands.
| | - Esrath Santha
- Division of Protein Expression and Modification, New England Biolabs, Ipswich, MA, 01938, USA
| | - Anna-Janina Behrens
- Division of Protein Expression and Modification, New England Biolabs, Ipswich, MA, 01938, USA
| | - D Linh Nguyen
- Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Mehul B Ganatra
- Division of Protein Expression and Modification, New England Biolabs, Ipswich, MA, 01938, USA
| | - Christopher H Taron
- Division of Protein Expression and Modification, New England Biolabs, Ipswich, MA, 01938, USA
| | - Vishal Khatri
- Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, Rockford, IL, USA
| | - Ramaswamy Kalyanasundaram
- Department of Biomedical Sciences, University of Illinois College of Medicine at Rockford, Rockford, IL, USA
| | - Angela van Diepen
- Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Cornelis H Hokke
- Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Jeremy M Foster
- Division of Protein Expression and Modification, New England Biolabs, Ipswich, MA, 01938, USA.
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26
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Kubo N, Yazawa S, Yokobori T, Sano R, Eguchi H, Kobayashi S, Akita H, Mitsufuji S, Yamashita YI, Nakao Y, Fujii T, Okumura T, Shibuya K, Hoshino Y, Yamada S, Hayashi M, Shimokawa M, Shirabe K. The malignant potential of pancreatic intraductal papillary mucinous neoplasm is reflected in expression levels of fucosylated glycans in α 1 -acid glycoprotein. JOURNAL OF HEPATO-BILIARY-PANCREATIC SCIENCES 2022; 30:503-513. [PMID: 35776060 DOI: 10.1002/jhbp.1208] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Revised: 05/07/2022] [Accepted: 06/03/2022] [Indexed: 11/08/2022]
Abstract
BACKGROUND Pancreatic intraductal papillary mucinous neoplasm (IPMN) involves multiple histopathological stages from benign to malignant lesions. Further, a biomarker to diagnose the malignant IPMN (IPMC) is clinically relevant. Recently, we found that serum fucosylated α1 -acid glycoprotein (fAGP) level markedly elevated along with disease progression in large cohorts of patients with various cancers. METHODS The fAGP level was retrospectively analyzed in preoperative sera from 109 patients with IPMN, and the clinical relevance of fAGP was compared with currently available predictors as standard. RESULTS The fAGP level in IPMC was found to be significantly higher than in benign IPMN (P=0.0012). At a cutoff value of 27.04 U/μg, its sensitivity, specificity, and accuracy for IPMC were determined to be 83.61, 65.96 and 75.93%, respectively. Multivariate analyses revealed that the fAGP level was the only independent risk factor for predicting IPMC. Additionally, a combination of the fAGP level and 18 F-fluorodeoxyglucose uptake on the PET/CT imaging in the lesions seemed to offer the best diagnosis of IPMN. Accordingly, 27 of the 28 patients who were positive in both tests had IPMC, while patients who are negative had benign IPMN. CONCLUSIONS The fAGP level appeared to be a relevant biomarker for malignant potential of IPMN.
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Affiliation(s)
- Norio Kubo
- Division of Hepatobiliary and Pancreatic Surgery, Department of General Surgical Science, Graduate School of Medicine, Gunma University, Maebashi, Japan
| | - Shin Yazawa
- Division of Hepatobiliary and Pancreatic Surgery, Department of General Surgical Science, Graduate School of Medicine, Gunma University, Maebashi, Japan
| | - Takehiko Yokobori
- Department of Innovative Cancer Immunotherapy, Graduate School of Medicine, Gunma University, Maebashi, Japan
| | - Rie Sano
- Department of Legal Medicine, Graduate School of Medicine, Gunma University, Maebashi, Japan
| | - Hidetoshi Eguchi
- Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Suita, Japan
| | - Shogo Kobayashi
- Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Suita, Japan
| | - Hirofumi Akita
- Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Suita, Japan
| | - Suguru Mitsufuji
- Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Suita, Japan
| | - Yo-Ichi Yamashita
- Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Yosuke Nakao
- Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Tsutomu Fujii
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama, Japan
| | - Tomoyuki Okumura
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama, Japan
| | - Kazuto Shibuya
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama, Japan
| | - Yui Hoshino
- Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama, Japan
| | - Suguru Yamada
- Department of Gastroenterological Surgery (Surgery II), Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Masamichi Hayashi
- Department of Gastroenterological Surgery (Surgery II), Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Mototsugu Shimokawa
- Department of Biostatistics, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan
| | - Ken Shirabe
- Division of Hepatobiliary and Pancreatic Surgery, Department of General Surgical Science, Graduate School of Medicine, Gunma University, Maebashi, Japan
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27
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Tijardović M, Štambuk T, Juszczak A, Keser T, Gasperikova D, Novokmet M, Tjora E, Pape Medvidović E, Stanik J, Rasmus Njølstad P, Lauc G, Owen KR, Gornik O. Fucosylated AGP glycopeptides as biomarkers of HNF1A-Maturity onset diabetes of the young. Diabetes Res Clin Pract 2022; 185:109226. [PMID: 35122907 DOI: 10.1016/j.diabres.2022.109226] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/09/2021] [Revised: 01/19/2022] [Accepted: 01/28/2022] [Indexed: 11/16/2022]
Abstract
AIMS We previously demonstrated that antennary fucosylated N-glycans on plasma proteins are regulated by HNF1A and can identify cases of Maturity-Onset Diabetes of the Young caused by HNF1A variants (HNF1A-MODY). Based on literature data, we further postulated that N-glycans with best diagnostic value mostly originate from alpha-1-acid glycoprotein (AGP). In this study we analyzed fucosylation of AGP in subjects with HNF1A-MODY and other types of diabetes aiming to evaluate its diagnostic potential. METHODS A recently developed LC-MS method for AGP N-glycopeptide analysis was utilized in two independent cohorts: a) 466 subjects with different diabetes subtypes to test the fucosylation differences, b) 98 selected individuals to test the discriminative potential for pathogenic HNF1A variants. RESULTS Our results showed significant reduction in AGP fucosylation associated to HNF1A-MODY when compared to other diabetes subtypes. Additionally, ROC curve analysis confirmed significant discriminatory potential of individual fucosylated AGP glycopeptides, where the best performing glycopeptide had an AUC of 0.94 (95% CI 0.90-0.99). CONCLUSIONS A glycopeptide based diagnostic tool would be beneficial for patient stratification by providing information about the functionality of HNF1A. It could assist the interpretation of DNA sequencing results and be a useful addition to the differential diagnostic process.
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Affiliation(s)
- Marko Tijardović
- Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia
| | | | - Agata Juszczak
- Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, University of Oxford, Oxford, UK; Oxford NIHR Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, John Radcliffe Hospital, Oxford, UK
| | - Toma Keser
- Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia
| | - Daniela Gasperikova
- Department of Metabolic Disorders, Institute of Experimental Endocrinology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia
| | | | - Erling Tjora
- Children and Youth Clinic, Haukeland University Hospital, Bergen, Norway
| | - Edita Pape Medvidović
- Vuk Vrhovac University Clinic for Diabetes, Endocrinology and Metabolic Diseases, Merkur University Hospital, Zagreb University School of Medicine, Zagreb, Croatia
| | - Juraj Stanik
- Department of Metabolic Disorders, Institute of Experimental Endocrinology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia; Department of Pediatrics, Medical Faculty of Comenius University and National Institute for Children's Diseases, Bratislava, Slovakia
| | - Pål Rasmus Njølstad
- Children and Youth Clinic, Haukeland University Hospital, Bergen, Norway; Center for Diabetes Research, Department of Clinical Science, University of Bergen, Bergen, Norway
| | - Gordan Lauc
- Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia; Genos Glycoscience Research Laboratory, Zagreb, Croatia
| | - Katharine R Owen
- Oxford Centre for Diabetes, Endocrinology and Metabolism, Churchill Hospital, University of Oxford, Oxford, UK; Oxford NIHR Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, John Radcliffe Hospital, Oxford, UK
| | - Olga Gornik
- Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia.
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Zeng W, Zheng S, Su T, Cheng J, Mao Y, Zhong Y, Liu Y, Chen J, Zhao W, Lin T, Liu F, Li G, Yang H, Zhang Y. Comparative N-Glycoproteomics Analysis of Clinical Samples Via Different Mass Spectrometry Dissociation Methods. Front Chem 2022; 10:839470. [PMID: 35281567 PMCID: PMC8907888 DOI: 10.3389/fchem.2022.839470] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2021] [Accepted: 01/20/2022] [Indexed: 11/13/2022] Open
Abstract
Site-specific N-glycosylation characterization requires intact N-glycopeptide analysis based on suitable tandem mass spectrometry (MS/MS) method. Electron-transfer/higher-energy collisional dissociation (EThcD), stepped collision energy/higher-energy collisional dissociation (sceHCD), higher-energy collisional dissociation-product-dependent electron-transfer dissociation (HCD-pd-ETD), and a hybrid mass spectrometry fragmentation method EThcD-sceHCD have emerged as valuable approaches for glycoprotein analysis. However, each of them incurs some compromise, necessitating the systematic performance comparisons when applied to the analysis of complex clinical samples (e.g., plasma, urine, cells, and tissues). Herein, we compared the performance of EThcD-sceHCD with those previous approaches (EThcD, sceHCD, HCD-pd-ETD, and sceHCD-pd-ETD) in the intact N-glycopeptide analysis, and determined its applicability for clinical N-glycoproteomic study. The intact N-glycopeptides of distinct samples, namely, plasma from prostate cancer (PCa) patients, urine from immunoglobulin A nephropathy (IgAN) patients, human hepatocarcinoma cell line (HepG2), and thyroid tissues from thyroid cancer (TC) patients were analyzed by these methods. We found that EThcD-sceHCD outperformed other methods in the balance of depth and accuracy of intact N-glycopeptide identification, and sceHCD and EThcD-sceHCD have good complementarity. EThcD-sceHCD holds great potential for biomarker discovery from clinical samples.
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Affiliation(s)
- Wenjuan Zeng
- Institutes for Systems Genetics, National Health Commission (NHC) Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - Shanshan Zheng
- Institutes for Systems Genetics, National Health Commission (NHC) Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - Tao Su
- Institutes for Systems Genetics, National Health Commission (NHC) Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - Jiahan Cheng
- Department of Thoracic Surgery, Institute of Thoracic Oncology, West China Hospital, Sichuan University, Chengdu, China
| | - Yonghong Mao
- Department of Thoracic Surgery, Institute of Thoracic Oncology, West China Hospital, Sichuan University, Chengdu, China
| | - Yi Zhong
- Institutes for Systems Genetics, National Health Commission (NHC) Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - Yueqiu Liu
- Institutes for Systems Genetics, National Health Commission (NHC) Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - Jianhai Chen
- Institutes for Systems Genetics, National Health Commission (NHC) Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
| | - Wanjun Zhao
- Department of Thyroid Surgery, West China Hospital, Sichuan University, Chengdu, China
| | - Tianhai Lin
- Department of Urology, Institute of Urology, West China Hospital, Sichuan University, Chengdu, China
| | - Fang Liu
- Division of Nephrology, West China Hospital, Sichuan University, Chengdu, China
| | - Guisen Li
- Renal Department and Institute of Nephrology, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Sichuan Clinical Research Center for Kidney Diseases, Chengdu, China
| | - Hao Yang
- Institutes for Systems Genetics, National Health Commission (NHC) Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
- *Correspondence: Hao Yang, ; Yong Zhang,
| | - Yong Zhang
- Institutes for Systems Genetics, National Health Commission (NHC) Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu, China
- *Correspondence: Hao Yang, ; Yong Zhang,
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29
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Fang P, Ji Y, Oellerich T, Urlaub H, Pan KT. Strategies for Proteome-Wide Quantification of Glycosylation Macro- and Micro-Heterogeneity. Int J Mol Sci 2022; 23:ijms23031609. [PMID: 35163546 PMCID: PMC8835892 DOI: 10.3390/ijms23031609] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2021] [Revised: 01/26/2022] [Accepted: 01/27/2022] [Indexed: 12/03/2022] Open
Abstract
Protein glycosylation governs key physiological and pathological processes in human cells. Aberrant glycosylation is thus closely associated with disease progression. Mass spectrometry (MS)-based glycoproteomics has emerged as an indispensable tool for investigating glycosylation changes in biological samples with high sensitivity. Following rapid improvements in methodologies for reliable intact glycopeptide identification, site-specific quantification of glycopeptide macro- and micro-heterogeneity at the proteome scale has become an urgent need for exploring glycosylation regulations. Here, we summarize recent advances in N- and O-linked glycoproteomic quantification strategies and discuss their limitations. We further describe a strategy to propagate MS data for multilayered glycopeptide quantification, enabling a more comprehensive examination of global and site-specific glycosylation changes. Altogether, we show how quantitative glycoproteomics methods explore glycosylation regulation in human diseases and promote the discovery of biomarkers and therapeutic targets.
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Affiliation(s)
- Pan Fang
- Department of Biochemistry and Molecular Biology, School of Biology & Basic Medical Sciences, Suzhou Medical College of Soochow University, Suzhou 215123, China;
| | - Yanlong Ji
- Bioanalytical Mass Spectrometry Group, Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany;
- Hematology/Oncology, Department of Medicine II, Johann Wolfgang Goethe University, 60590 Frankfurt am Main, Germany;
- Frankfurt Cancer Institute, Johann Wolfgang Goethe University, 60596 Frankfurt am Main, Germany
| | - Thomas Oellerich
- Hematology/Oncology, Department of Medicine II, Johann Wolfgang Goethe University, 60590 Frankfurt am Main, Germany;
- Frankfurt Cancer Institute, Johann Wolfgang Goethe University, 60596 Frankfurt am Main, Germany
- German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
| | - Henning Urlaub
- Bioanalytical Mass Spectrometry Group, Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany;
- Institute of Clinical Chemistry, University Medical Center Göttingen, 37075 Göttingen, Germany
- Correspondence: (H.U.); (K.-T.P.)
| | - Kuan-Ting Pan
- Hematology/Oncology, Department of Medicine II, Johann Wolfgang Goethe University, 60590 Frankfurt am Main, Germany;
- Frankfurt Cancer Institute, Johann Wolfgang Goethe University, 60596 Frankfurt am Main, Germany
- Correspondence: (H.U.); (K.-T.P.)
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30
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Bai H, Zhang B, Cheng X, Liu J, Wang X, Qin W, Zhang M. Synthesis of zwitterionic polymer modified graphene oxide for hydrophilic enrichment of N-glycopeptides from urine of healthy subjects and patients with lung adenocarcinoma. Talanta 2022; 237:122938. [PMID: 34736669 DOI: 10.1016/j.talanta.2021.122938] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2021] [Revised: 09/26/2021] [Accepted: 10/05/2021] [Indexed: 10/20/2022]
Abstract
As one of the most common and important post-translational modifications, protein N-glycosylation plays essential roles in many biological processes and have long been considered closely correlated with the occurrence and progression of multiple diseases. Systematic characterization of these disease-related protein N-glycosylation is one of the most convenient ways for new diagnostic biomarker and therapeutic drug target discovering. However, the biological samples are extremely complex and the abundance of N-glycoproteins are especially low, which make highly efficient N-glycoprotein/glycopeptide enrichment before mass spectrometry analysis a prerequisite. In this work, a new type of hydrophilic material (GO-pDMAPS) was prepared by in situ growth of linear zwitterionic polymer chains on the surface of GO and it was successfully applied for N-glycopeptide enrichment from human urine. Due to the excellent hydrophilicity and the facilitate interactions between this GO-pDMAPS and the targets, a total of 1426 N-glycosylated sites corresponding to 766 N-glycoproteins as well as 790 N-glycosylation sites corresponding to 470 N-glycoproteins were enriched and identified from urine of healthy subjects and patients with lung adenocarcinoma, respectively. Among which, 27 N-glycoproteins were expressed exclusively and 4 N-glycoproteins were upregulated at least 3 times comparing with the healthy group, demonstrating the tremendous potential of this new hydrophilic material for large scale and in depth N-glycoproteome research.
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Affiliation(s)
- Haihong Bai
- Clinical Laboratory Medicine, Beijing Key Laboratory of Urinary Cellular Molecular Diagnostics, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, PR China; Phase Ⅰ Clinical Trial Center, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, PR China
| | - Baoying Zhang
- Phase Ⅰ Clinical Trial Center, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, PR China; State Key Laboratory of Proteomics, National Center for Protein Sciences Beijing, Beijing Institute of Lifeomics, Beijing Proteome Research Center, Beijing, 102206, PR China
| | - Xiaoqiang Cheng
- Phase Ⅰ Clinical Trial Center, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, PR China
| | - Ju Liu
- Phase Ⅰ Clinical Trial Center, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, PR China
| | - Xinghe Wang
- Phase Ⅰ Clinical Trial Center, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, PR China
| | - Weijie Qin
- State Key Laboratory of Proteomics, National Center for Protein Sciences Beijing, Beijing Institute of Lifeomics, Beijing Proteome Research Center, Beijing, 102206, PR China
| | - Man Zhang
- Clinical Laboratory Medicine, Beijing Key Laboratory of Urinary Cellular Molecular Diagnostics, Beijing Shijitan Hospital, Capital Medical University, Beijing, 100038, PR China.
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Abstract
In this editorial, the roles of orosomucoid (ORM) in the diagnoses and follow-up assessments of both nonneoplastic diseases and liver tumors are discussed with respect to the publication by Zhu et al presented in the previous issue of World Journal of Gastroenterology (2020; 26(8): 840-817). ORM, or alpha-1 acid glycoprotein (AGP), is an acute-phase protein that constitutes 1% to 3% of plasma proteins in humans and is mainly synthesized in the liver. ORM exists in serum as two variants: ORM1 and ORM2. Although the variants share 89.6% sequence identity and have similar biological properties, ORM1 constitutes the main component of serum ORM. An interesting feature of ORM is that its biological effects differ according to variations in glycosylation patterns. This variable feature makes ORM an attractive target for diagnosing and monitoring many diseases, including those of the liver. Recent findings suggest that a sharp decrease in ORM level is an important marker for HBV-associated acute liver failure (ALF), and ORM1 plays an important role in liver regeneration. In viral hepatitis, increases in both ORM and its fucosylated forms and the correlation of these increases with fibrosis progression suggest that this glycoprotein can be used with other markers as a noninvasive method in the follow-up assessment of diseases. In addition, similar findings regarding the level of the asialylated form of ORM, called asialo-AGP (AsAGP), have been reported in a follow-up assessment of fibrosis in chronic liver disease. An increase in ORM in serum has also been shown to improve hepatocellular carcinoma (HCC) diagnosis performance when combined with other markers. In addition, determination of the ORM level has been useful in the diagnosis of HCC with AFP concentrations less than 500 ng/mL. For monitoring patients with AFP-negative HCC, a unique trifucosylated tetra-antennary glycan of ORM may also be used as a new potential marker. The fact that there are very few studies investigating the expression of this glycoprotein and its variants in liver tissues constitutes a potential limitation, especially in terms of revealing all the effects of ORM on carcinogenesis and tumor behavior. Current findings indicate that ORM2 expression is decreased in tumors, and this is related to the aggressive course of the disease. Parallel to this finding, in HCC cell lines, ORM2 decreases HCC cell migration and invasion, supporting reports of its tumor suppressor role. In conclusion, the levels of ORM and its different glycosylated variants are promising additional biomarkers for identifying ALF, for monitoring fibrosis in viral hepatitis, and for diagnosing early HCC. Although there is evidence that the loss of ORM2 expression in HCC is associated with poor prognosis, further studies are needed to support these findings. Additionally, investigations of ORM expression in borderline dysplastic nodules and hepatocellular adenomas, which pose diagnostic problems in the differential diagnosis of HCC, especially in biopsy samples, may shed light on whether ORM can be used in histopathological differential diagnosis.
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Affiliation(s)
- Gulsum Ozlem Elpek
- Department of Pathology, Akdeniz University Medical School, Antalya 07070, Turkey
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32
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Cvetko A, Mangino M, Tijardović M, Kifer D, Falchi M, Keser T, Perola M, Spector TD, Lauc G, Menni C, Gornik O. Plasma N-glycome shows continuous deterioration as the diagnosis of insulin resistance approaches. BMJ Open Diabetes Res Care 2021; 9:9/1/e002263. [PMID: 34518155 PMCID: PMC8438737 DOI: 10.1136/bmjdrc-2021-002263] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/11/2021] [Accepted: 08/22/2021] [Indexed: 12/12/2022] Open
Abstract
INTRODUCTION Prediction of type 2 diabetes mellitus (T2DM) and its preceding factors, such as insulin resistance (IR), is of great importance as it may allow delay or prevention of onset of the disease. Plasma protein N-glycome has emerged as a promising predictive biomarker. In a prospective longitudinal study, we included patients with a first diagnosis of impaired glucose metabolism (IR or T2DM) to investigate the N-glycosylation's predictive value years before diabetes development. RESEARCH DESIGN AND METHODS Plasma protein N-glycome was profiled by hydrophilic interaction ultra-performance liquid chromatography in 534 TwinsUK participants free from disease at baseline. This included 89 participants with incident diagnosis of IR or T2DM during the follow-up period (7.14±3.04 years) whose last sample prior to diagnosis was compared using general linear regression with 445 age-matched unrelated controls. Findings were replicated in an independent cohort. Changes in N-glycome have also been presented in connection with time to diagnosis. RESULTS Eight groups of plasma N-glycans were different between incident IR or T2DM cases and controls (p<0.05) after adjusting for multiple testing using Benjamini-Hochberg correction. These differences were noticeable up to 10 years prior to diagnosis and are changing continuously as becoming more expressed toward the diagnosis. The prediction model was built using significant glycan traits, displaying a discriminative performance with an area under the receiver operating characteristic curve of 0.77. CONCLUSIONS In addition to previous studies, we showed the diagnostic potential of plasma N-glycome in the prediction of both IR and T2DM development years before the clinical manifestation and indicated the continuous deterioration of N-glycome toward the diagnosis.
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Affiliation(s)
- Ana Cvetko
- University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
| | - Massimo Mangino
- Department of Twin Research and Genetic Epidemiology, King's College London, London, UK
- NIHR Biomedical Research Centre at Guy's and St Thomas' Foundation Trust, London, UK
| | - Marko Tijardović
- University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
| | - Domagoj Kifer
- University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
| | - Mario Falchi
- Department of Twin Research and Genetic Epidemiology, King's College London, London, UK
| | - Toma Keser
- University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
| | - Markus Perola
- National Institute for Health and Welfare, Helsinki, Finland
| | - Tim D Spector
- Department of Twin Research and Genetic Epidemiology, King's College London, London, UK
| | - Gordan Lauc
- University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
- Genos Glycoscience Research Laboratory, Zagreb, Croatia
| | - Cristina Menni
- Department of Twin Research and Genetic Epidemiology, King's College London, London, UK
| | - Olga Gornik
- University of Zagreb Faculty of Pharmacy and Biochemistry, Zagreb, Croatia
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Zhang L, Ma L, Li J, Lei J, Chen J, Yu C. VE-cadherin N-glycosylation modified by N-acetylglucosaminyltransferase V regulates VE-cadherin-β-catenin interaction and monocyte adhesion. Exp Physiol 2021; 106:1869-1877. [PMID: 34117813 DOI: 10.1113/ep089617] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Accepted: 06/10/2021] [Indexed: 11/08/2022]
Abstract
NEW FINDINGS What is the central question of this study? Inflammation-induced monocyte adhesion is the initiator of most vascular diseases. The underlying mechanisms that mediate monocyte adhesion remain to be clarified fully. What is the main finding and its importance? N-acetylglucosaminyltransferase V (GnT-V)-mediated N-glycosylation of VE-cadherin regulates the dissociation of the VE-cadherin-β-catenin complex to modulate monocyte adhesion, but GnT-V overexpression cannot rescue monocyte adhesion induced by interleukin-1β. This study clarified the molecular mechanism of VE-cadherin in regulating the monocyte adhesion process. ABSTRACT Monocyte adhesion is a crucial step in the initial stage of atherosclerosis, and dysfunction of VE-cadherin has been reported to be involved in this process. Our group previously found that VE-cadherin and its binding protein, β-catenin, were modified by sialylation, and the levels of sialylation were decreased in pro-inflammatory cytokine-treated human umbilical vein EA.hy926 cells. In this study, we confirmed that the sugar chains of VE-cadherin were modified by N-acetylglucosaminyltransferase V (GnT-V). We showed that the levels of GnT-V and β1,6-N-acetylglucosamine on the VE-cadherin were reduced in the presence of interleukin-1β, whereas the level of monocyte transendothelial migration was increased. Moreover, the interaction between VE-cadherin and β-catenin was increased, accompanied by an increased accumulation of degradative VE-cadherin and cytoplasmic β-catenin, indicating impairment of cell-cell junctions after interleukin-1β treatment. Furthermore, GnT-V short hairpin RNA and overexpression analysis confirmed that glycosylation of VE-cadherin was modified by GnT-V in EA.hy926 cells, which contributed to the monocyte-endothelial adhesion process. Taken together, these results suggest that the function of VE-cadherin in facilitating monocyte adhesion might result from the decreasing GnT-V expression and disorder of GnT-V-catalysed N-glycosylation. Our study clarified the molecular mechanism of VE-cadherin in regulation of the monocyte adhesion process and provided new insights into the post-transcriptional modifications of VE-cadherin.
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Affiliation(s)
- Lei Zhang
- College of Pharmacy, Chongqing Medical University, Chongqing, PR China.,Chongqing Key Laboratory for Pharmaceutical Metabolism Research, Chongqing, PR China
| | - Limei Ma
- College of Pharmacy, Chongqing Medical University, Chongqing, PR China.,Chongqing Key Laboratory for Pharmaceutical Metabolism Research, Chongqing, PR China
| | - Jiajia Li
- College of Pharmacy, Chongqing Medical University, Chongqing, PR China.,Chongqing Key Laboratory for Pharmaceutical Metabolism Research, Chongqing, PR China.,Department of Pharmacy, Chongqing Hechuan District People's Hospital, Chongqing, PR China
| | - Jin Lei
- College of Pharmacy, Chongqing Medical University, Chongqing, PR China.,Chongqing Key Laboratory for Pharmaceutical Metabolism Research, Chongqing, PR China
| | - Jun Chen
- College of Pharmacy, Chongqing Medical University, Chongqing, PR China.,Chongqing Key Laboratory for Pharmaceutical Metabolism Research, Chongqing, PR China
| | - Chao Yu
- College of Pharmacy, Chongqing Medical University, Chongqing, PR China.,Chongqing Key Laboratory for Pharmaceutical Metabolism Research, Chongqing, PR China
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Tabang DN, Ford M, Li L. Recent Advances in Mass Spectrometry-Based Glycomic and Glycoproteomic Studies of Pancreatic Diseases. Front Chem 2021; 9:707387. [PMID: 34368082 PMCID: PMC8342852 DOI: 10.3389/fchem.2021.707387] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2021] [Accepted: 07/12/2021] [Indexed: 12/14/2022] Open
Abstract
Modification of proteins by glycans plays a crucial role in mediating biological functions in both healthy and diseased states. Mass spectrometry (MS) has emerged as the most powerful tool for glycomic and glycoproteomic analyses advancing knowledge of many diseases. Such diseases include those of the pancreas which affect millions of people each year. In this review, recent advances in pancreatic disease research facilitated by MS-based glycomic and glycoproteomic studies will be examined with a focus on diabetes and pancreatic cancer. The last decade, and especially the last five years, has witnessed developments in both discovering new glycan or glycoprotein biomarkers and analyzing the links between glycans and disease pathology through MS-based studies. The strength of MS lies in the specificity and sensitivity of liquid chromatography-electrospray ionization MS for measuring a wide range of biomolecules from limited sample amounts from many sample types, greatly enhancing and accelerating the biomarker discovery process. Furthermore, imaging MS of glycans enabled by matrix-assisted laser desorption/ionization has proven useful in complementing histology and immunohistochemistry to monitor pancreatic disease progression. Advances in biological understanding and analytical techniques, as well as challenges and future directions for the field, will be discussed.
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Affiliation(s)
- Dylan Nicholas Tabang
- Department of Chemistry, University of Wisconsin-Madison, Madison, WI, United States
| | - Megan Ford
- Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, United States
| | - Lingjun Li
- Department of Chemistry, University of Wisconsin-Madison, Madison, WI, United States.,School of Pharmacy, University of Wisconsin-Madison, Madison, WI, United States
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35
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Ma J, Sun S, Ni C, Li L, Xia J, Li H, Song H, Heng X, Hu D, Li Y. Proteomic analysis of overweight/obesity and related abnormal glucose and lipid metabolism caused by phlegm-dampness retention. JOURNAL OF TRADITIONAL CHINESE MEDICAL SCIENCES 2021. [DOI: 10.1016/j.jtcms.2021.07.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
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36
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Ruiz M. Into the Labyrinth of the Lipocalin α1-Acid Glycoprotein. Front Physiol 2021; 12:686251. [PMID: 34168570 PMCID: PMC8217824 DOI: 10.3389/fphys.2021.686251] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Accepted: 05/17/2021] [Indexed: 12/28/2022] Open
Abstract
α1-acid glycoprotein (AGP), also known as Orosomucoid (ORM), belongs to the Lipocalin protein family and it is well-known for being a positive acute-phase protein. AGP is mostly found in plasma, with the liver as main contributor, but it is also expressed in other tissues such as the brain or the adipose tissue. Despite the vast literature on AGP, the physiological functions of the protein remain to be elucidated. A large number of activities mostly related to protection and immune system modulation have been described. Recently created AGP-knockout models have suggested novel physiological roles of AGP, including regulation of metabolism. AGP has an outstanding ability to efficiently bind endogenous and exogenous small molecules that together with the complex and variable glycosylation patterns, determine AGP functions. This review summarizes and discusses the recent findings on AGP structure (including glycans), ligand-binding ability, regulation, and physiological functions of AGP. Moreover, this review explores possible molecular and functional connections between AGP and other members of the Lipocalin protein family.
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Affiliation(s)
- Mario Ruiz
- Department of Chemistry and Molecular Biology, University of Gothenburg, Gothenburg, Sweden
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