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High-throughput site-specific N-glycoproteomics reveals glyco-signatures for liver disease diagnosis.

ABSTRACT: The glycoproteome has emerged as a prominent target for screening biomarkers, as altered glycosylation is a hallmark of cancer cells. In this work, we incorporated tandem mass tag labeling into quantitative glycoproteomics by developing a chemical labeling-assisted complementary dissociation method for the multiplexed analysis of intact N-glycopeptides. Benefiting from the complementary nature of two different mass spectrometry dissociation methods for identification and multiplex labeling for quantification of intact N-glycopeptides, we conducted the most comprehensive site-specific and subclass-specific N-glycosylation profiling of human serum immunoglobulin G (IgG) to date. By analysing the serum of 90 human patients with varying severities of liver diseases, as well as healthy controls, we identified that the combination of IgG1-H3N5F1 and IgG4-H4N3 can be used for distinguishing between different stages of liver diseases. Finally, we used targeted parallel reaction monitoring to successfully validate the expression changes of glycosylation in liver diseases in a different sample cohort that included 45 serum samples.

SUBMITTER: Sun Z

PROVIDER: S-EPMC9985154 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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High-throughput site-specific <i>N</i>-glycoproteomics reveals glyco-signatures for liver disease diagnosis.

Sun Zhenyu Z Fu Bin B Wang Guoli G Zhang Lei L Xu Ruofan R Zhang Ying Y Lu Haojie H

National science review 20220405 1

The glycoproteome has emerged as a prominent target for screening biomarkers, as altered glycosylation is a hallmark of cancer cells. In this work, we incorporated tandem mass tag labeling into quantitative glycoproteomics by developing a chemical labeling-assisted complementary dissociation method for the multiplexed analysis of intact <i>N</i>-glycopeptides. Benefiting from the complementary nature of two different mass spectrometry dissociation methods for identification and multiplex labelin ...[more]

PMID: 36879659

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Project description:Glycosylation influences diverse life processes and changes in pathological processes. The glycoproteomic has been recognized as a hot area to screen markers as altered glycosylation is a universal feature of cancer cells. However, the high throughput screening of glycosylation markers from a large number of clinical samples faces greater technical challenges than peptides without or with other modifications. The most prominent problem in tandem mass tag-based high throughput quantification of intact glycopeptides is the inefficient cleavage of peptide backbone, glycosidic bonds and tandem mass tag concurrently to offer successful peptide and glycan composition determination as well as good quantification. Here, we managed to implement the tandem mass tag labeling into quantitative glycoproteomics by developing a chemical labeling-assisted complementary dissociation method for multiplexed analysis of intact N-glycopeptides. Chemical labeling of intact N-glycopeptides with charge-increasing reagent improved the efficiency of electron transfer dissociation (ETD) significantly for their identification and also increased the efficiency of higher-energy collision dissociation (HCD) in generating reporter ion for quantification, thereby capitalizing on the complementary nature of these two dissociation methods in quantification of intact N-glycopeptides. By using this new strategy, we investigated the site-specific glycosylation of IgG collected from 90 human serum including three stages of liver diseases, hepatitis B virus (HBV), cirrhosis (CIR), hepatocellular carcinoma (HCC) and healthy controls. As a result, 313 intact N-glycopeptides from IgG were identified in total, which represents the most comprehensive site-specific and subclass-specific N-glycosylation of human serum IgG to date. Quantitative glycoproteomics revealed that the combination of IgG1-H3N5F1 and IgG4-H3N4 displayed powerful prediction capability for distinguishing different stages of liver disease patients, which has potential for non-invasive monitoring and pre-stratiﬁcation of liver disease diagnosis. Moreover, we analyzed the human serum using this newly developed method and identified near 2000 intact N-glycopeptides from only 10 L serum and showed this method has applicability for quantification of intact N-glycopeptides in different studies.

Project description:IntroductionThe human plasma glycoproteome holds enormous potential to identify personalized biomarkers for diagnostics. Glycoproteomics has matured into a technology for plasma N-glycoproteome analysis but further evolution towards clinical applications depends on the clinical validity and understanding of protein- and site-specific glycosylation changes in disease.ObjectivesHere, we exploited the uniqueness of a patient cohort of genetic defects in well-defined glycosylation pathways to assess the clinical applicability of plasma N-glycoproteomics.MethodsComparative glycoproteomics was performed of blood plasma from 40 controls and 74 patients with 13 different genetic diseases that impact the protein N-glycosylation pathway. Baseline glycosylation in healthy individuals was compared to reference glycome and intact transferrin protein mass spectrometry data. Use of glycoproteomics data for biomarker discovery and sample stratification was evaluated by multivariate chemometrics and supervised machine learning. Clinical relevance of site-specific glycosylation changes were evaluated in the context of genetic defects that lead to distinct accumulation or loss of specific glycans. Integrated analysis of site-specific glycoproteome changes in disease was performed using chord diagrams and correlated with intact transferrin protein mass spectrometry data.ResultsGlycoproteomics identified 191 unique glycoforms from 58 unique peptide sequences of 34 plasma glycoproteins that span over 3 magnitudes of abundance in plasma. Chemometrics identified high-specificity biomarker signatures for each of the individual genetic defects with better stratification performance than the current diagnostic standard method. Bioinformatic analyses revealed site-specific glycosylation differences that could be explained by underlying glycobiology and protein-intrinsic factors.ConclusionOur work illustrates the strong potential of plasma glycoproteomics to significantly increase specificity of glycoprotein biomarkers with direct insights in site-specific glycosylation changes to better understand the glycobiological mechanisms underlying human disease.

Project description:BackgroundCD4+ T cells play critical roles in the pathogenesis of IgG4-related disease (IgG4-RD). The aim of this study was to investigate the TCR repertoire of peripheral blood CD4+ T cells in IgG4-RD.MethodsThe peripheral blood was collected from six healthy controls and eight IgG4-RD patients. TCR β-chain libraries of CD4+ T cells were constructed by 5'-rapid amplification of cDNA ends (5'-RACE) and sequenced by Illumina Miseq platform. The relative similarity of TCR repertoires between samples was evaluated according to the total frequencies of shared clonotypes (metric F), correlation of frequencies of shared clonotypes (metric R), and total number of shared clonotypes (metric D).ResultsThe clonal expansion and diversity of CD4+ T cell repertoire were comparable between healthy controls and IgG4-RD patients, while the proportion of expanded and coding degenerated clones, as an indicator of antigen-driven clonal expansion, was significantly higher in IgG4-RD patients. There was no significant difference in TRBV and TRBJ gene usage between healthy controls and IgG4-RD patients. The complementarity determining region 3 (CDR3) length distribution was skewed towards longer fragments in IgG4-RD. Visualization of relative similarity of TCR repertoires by multi-dimensional scaling analysis showed that TCR repertoires of IgG4-RD patients were separated from that of healthy controls in F and D metrics. We identified 11 IgG4-RD-specific CDR3 amino acid sequences that were expanded in at least 2 IgG4-RD patients, while not detected in healthy controls. According to TCR clonotype networks constructed by connecting all the CDR3 sequences with a Levenshtein distance of 1, 3 IgG4-RD-specific clusters were identified. We annotated the TCR sequences with known antigen specificity according to McPAS-TCR database and found that the frequencies of TCR sequences associated with each disease or immune function were comparable between healthy controls and IgG4-RD patients.ConclusionAccording to our study of CD4+ T cells from eight IgG4-RD patients, TCR repertoires of IgG4-RD patients were different from that of healthy controls in the proportion of expanded and coding degenerated clones and CDR3 length distribution. In addition, IgG4-RD-specific TCR sequences and clusters were identified in our study.

Dataset Information

High-throughput site-specific N-glycoproteomics reveals glyco-signatures for liver disease diagnosis.

Publications

High-throughput site-specific <i>N</i>-glycoproteomics reveals glyco-signatures for liver disease diagnosis.

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