Quantitative analysis of core fucosylation of serum proteins in liver diseases by LC-MS-MRM.
ABSTRACT: Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. BIOLOGICAL SIGNIFICANCE: We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and selective LC-MS-MRM method. Despite an overall increase in core fucosylation of many of the glycoproteins that we examined, core fucosylation changed in a protein- and site-specific manner. Moreover, increased and decreased fucosylation was observed on different N-glycopeptides of the same protein. Altered core fucosylation of N-glycopeptides might be used as an alternative serologic assay for the evaluation of fibrotic liver disease.
Project description:Quantitative analysis of site specific glycoforms of proteins is technically challenging but highly desirable; resolution of the fucosylated glycoforms is of particular interest due to their biological importance. In this study, we developed a sensitive and specific LC-MS-MRM quantification method that distinguishes the outer arm and core fucosylated configurations of the N-glycopeptides. We take advantage of limited fragmentation of the glycopeptides at low collision energy CID to produce linkage-specific Y-ions. We select these informative ions as MRM transitions for the quantification of the outer arm and total fucosylation of 12 fucosylated glycoforms of 9 glycopeptides in 7 plasma proteins. Our workflow showed improved sensitivity and specificity of quantification of the glycopeptides compared to oxonium ion transitions which allowed us to quantify the glycoforms directly in plasma or serum without fractionation of the samples or glycopeptide enrichment. A pilot study of fucosylation in liver cirrhosis of the HCV and NASH etiologies confirms the quantitative capabilities of the method and shows that liver cirrhosis is consistently associated with increased outer arm fucosylation of majority of the analyzed proteins. The results show that the outer arm fucosylation of the A2G2F1 glycoform of the VDKDLQSLEDILHQVENK peptide of fibrinogen increases greater than 10-fold in the HCV and NASH patients compared to healthy controls.
Project description:Enhanced fucosylation has been suggested as a marker for serologic monitoring of liver disease and hepatocellular carcinoma (HCC). We present a workflow for quantitative site-specific analysis of fucosylation and apply it to a comparison of hemopexin (HPX) and complement factor H (CFH), two liver-secreted glycoproteins, in healthy individuals and patients with liver cirrhosis and HCC. Label-free LC-MS quantification of glycopeptides derived from these purified glycoproteins was performed on pooled samples (2 pools/group, 5 samples/pool) and complemented by glycosidase assisted analysis using sialidase and endoglycosidase F2/F3, respectively, to improve resolution of glycoforms. Our analysis, presented as relative abundance of individual fucosylated glycoforms normalized to the level of their nonfucosylated counterparts, revealed a consistent increase in fucosylation in liver disease with significant site- and protein-specific differences. We have observed the highest microheterogeneity of glycoforms at the N187 site of HPX, absence of core fucosylation at N882 and N911 sites of CFH, or a higher degree of core fucosylation in CFH compared to HPX, but we did not identify changes differentiating HCC from matched cirrhosis samples. Glycosidase assisted LC-MS-MRM analysis of individual patient samples prepared by a simplified protocol confirmed the quantitative differences. Transitions specific to outer arm fucose document a disease-associated increase in outer arm fucose on both bi- and triantennary glycans at the N187 site of HPX. Further verification is needed to confirm that enhanced fucosylation of HPX and CFH may serve as an indicator of premalignant liver disease. The analytical strategy can be readily adapted to analysis of other proteins in the appropriate disease context.
Project description:A mass spectrometry-based methodology has been developed to study changes in core-fucosylation of serum ceruloplasmin that are site-specific between cirrhosis and hepatocellular carcinoma (HCC). The serum samples studied for these changes were from patients affected by cirrhosis or HCC with different etiologies, including alcohol, hepatitis B virus, or hepatitis C virus. The methods involved trypsin digestion of ceruloplasmin into peptides followed by Endo F3 digestion, which removed most of the glycan structure while retaining the innermost N-acetylglucosamine (GlcNAc) and/or core-fucose bound to the peptide. This procedure simplified the structures for further analysis by mass spectrometry, where four core-fucosylated sites (sites 138, 358, 397, and 762) were detected in ceruloplasmin. The core-fucosylation ratio of three of these sites increased significantly in alcohol-related HCC samples (sample size = 24) compared to that in alcohol-related cirrhosis samples (sample size = 18), with the highest AUC value of 0.838 at site 138. When combining the core-fucosylation ratio of site 138 in ceruloplasmin and the alpha-fetoprotein (AFP) value, the AUC value increased to 0.954 (ORsite138 = 12.26, p = 0.017; ORAFP = 3.64, p = 0.022), which was markedly improved compared to that of AFP (AUC = 0.867) (LR test p = 0.0002) alone. However, in HBV- or HCV-related liver diseases, no significant site-specific change in core-fucosylation of ceruloplasmin was observed between HCC and cirrhosis.
Project description:Protein glycosylation is known to be involved in biological progresses such as cell recognition, growth, differentiation, and apoptosis. Fucosylation of glycoproteins plays an important role for structural stability and function of N-linked glycoproteins. Although many of biological and clinical studies of protein fucosylation by fucosyltransferases has been reported, structural classification of fucosylated N-glycoproteins such as core or outer isoforms remains a challenge. Here, we report for the first time the classification of N-glycopeptides as core- and outer-fucosylated types using tandem mass spectrometry (MS/MS) and machine learning algorithms such as the deep neural network (DNN) and support vector machine (SVM). Training and test sets of more than 800 MS/MS spectra of N-glycopeptides from the immunoglobulin gamma and alpha 1-acid-glycoprotein standards were selected for classification of the fucosylation types using supervised learning models. The best-performing model had an accuracy of more than 99% against manual characterization and area under the curve values greater than 0.99, which were calculated by probability scores from target and decoy datasets. Finally, this model was applied to classify fucosylated N-glycoproteins from human plasma. A total of 82N-glycopeptides, with 54 core-, 24 outer-, and 4 dual-fucosylation types derived from 54 glycoproteins, were commonly classified as the same type in both the DNN and SVM. Specifically, outer fucosylation was dominant in tri- and tetra-antennary N-glycopeptides, while core fucosylation was dominant in the mono-, bi-antennary and hybrid types of N-glycoproteins in human plasma. Thus, the machine learning methods can be combined with MS/MS to distinguish between different isoforms of fucosylated N-glycopeptides.
Project description:Core fucosylation of N-linked glycoproteins has been linked to the functions of glycoproteins in physiological and pathological processes. However, quantitative characterization of core fucosylation remains challenging due to the complexity and heterogeneity of N-linked glycosylation. Here we report a mass spectrometry-based method that employs sequential treatment of intact glycopeptides with enzymes (STAGE) to analyze site-specific core fucosylation of glycoproteins. The STAGE method utilizes Endo F3 followed by PNGase F treatment to generate mass signatures for glycosites that are formerly modified by core fucosylated N-linked glycans. We benchmark the STAGE method and use it to characterize site specific core fucosylation of glycoproteins from human hepatocellular carcinoma and pancreatic ductal adenocarcinoma, resulting in the identification of 1130 and 782 core fucosylated glycosites, respectively. These results indicate that our STAGE method enables quantitative characterization of core fucosylation events from complex protein mixtures, which may benefit our understanding of core fucosylation functions in various diseases.
Project description:Cirrhosis of the liver is associated with increased fucosylation of proteins in the plasma. We describe a data-independent (DIA) strategy for comparative analysis of the site-specific glycoforms of plasma glycoproteins. A library of 161 glycoforms of 25 N-glycopeptides was established by data-dependent LC-MS/MS analysis of a tryptic digest of 14 human protein groups retained on a multiple affinity removal column. The collision-induced dissociation conditions were adjusted to maximize the yield of selective Y-ions which were quantified by a data-independent mass spectrometry workflow using a 10-Da acquisition window. Using this workflow, we quantified 125 glycoforms of 25 glycopeptides, covering 10 of the 14 proteins, without any further glycopeptide enrichment. Comparison of the proteins in the plasma of healthy controls and cirrhotic patients shows an average 1.5-fold increase in the fucosylation of bi-antennary glycoforms and 3-fold increase in the fucosylation of tri- and tetra- antennary glycoforms. These results show that the adjusted glycopeptide DIA workflow using soft collision-induced fragmentation of glycopeptides is suitable for site-specific analysis of protein glycosylation in complex mixtures of analytes without glycopeptide enrichment.
Project description:A MS-based methodology has been developed for analysis of core-fucosylated versus antennary-fucosylated glycosites in glycoproteins. This procedure is applied to the glycoprotein alpha-1-antitrypsin (A1AT), which contains both core- and antennary-fucosylated glycosites. The workflow involves digestion of intact glycoproteins into glycopeptides, followed by double digestion with sialidase and galactosidase. The resulting glycopeptides with truncated glycans were separated using an off-line HILIC (hydrophilic interaction liquid chromatography) separation where multiple fractions were collected at various time intervals. The glycopeptides in each fraction were treated with PNGase F and then divided into halves. One half of the sample was applied for peptide identification while the other half was processed for glycan analysis by derivatizing with a meladrazine reagent followed by MS analysis. This procedure provided site-specific identification of glycosylation sites and the ability to distinguish core fucosylation and antennary fucosylation via a double digestion and a mass profile scan. Both core and antennary fucosylation are shown to be present on various glycosites in A1AT.
Project description:N-glycosylation alteration has been reported in liver diseases. Characterizing N-glycopeptides that correspond to N-glycan structure with specific site information enables better understanding of the molecular pathogenesis of liver damage and cancer. Here, unbiased quantification of N-glycopeptides of a cluster of serum glycoproteins with 40-55 kDa molecular weight (40-kDa band) was investigated in hepatitis B virus (HBV)-related liver diseases. We used an N-glycopeptide method based on 18O/16O C-terminal labeling to obtain 82 comparisons of serum from patients with HBV-related hepatocellular carcinoma (HCC) and liver cirrhosis (LC). Then, multiple reaction monitoring (MRM) was performed to quantify N-glycopeptide relative to the protein content, especially in the healthy donor-HBV-LC-HCC cascade. TPLTAN 205ITK (H5N5S1F1) and (H5N4S2F1) corresponding to the glycopeptides of IgA2 were significantly elevated in serum from patients with HBV infection and even higher in HBV-related LC patients, as compared with healthy donor. In contrast, the two glycopeptides of IgA2 fell back down in HBV-related HCC patients. In addition, the variation in the abundance of two glycopeptides was not caused by its protein concentration. The altered N-glycopeptides might be part of a unique glycan signature indicating an IgA-mediated mechanism and providing potential diagnostic clues in HBV-related liver diseases.
Project description:Core fucosylation of N-glycoproteins plays a crucial role in modulating the biological functions of glycoproteins. Yet, the synthesis of structurally well-defined, core-fucosylated glycoproteins remains a challenging task due to the complexity in multistep chemical synthesis or the inability of the biosynthetic ?1,6-fucosyltransferase (FUT8) to directly fucosylate full-size mature N-glycans in a chemoenzymatic approach. We report in this paper the design and generation of potential ?1,6-fucosynthase and fucoligase for direct core fucosylation of intact N-glycoproteins. We found that mutation at the nucleophilic residue (D200) did not provide a typical glycosynthase from this bacterial enzyme, but several mutants with mutation at the general acid/base residue E274 of the Lactobacillus casei ?1,6-fucosidase, including E274A, E274S, and E274G, acted as efficient glycoligases that could fucosylate a wide variety of complex N-glycopeptides and intact glycoproteins by using ?-fucosyl fluoride as a simple donor substrate. Studies on the substrate specificity revealed that the ?1,6-fucosidase mutants could introduce an ?1,6-fucose moiety specifically at the Asn-linked GlcNAc moiety not only to GlcNAc-peptide but also to high-mannose and complex-type N-glycans in the context of N-glycopeptides, N-glycoproteins, and intact antibodies. This discovery opens a new avenue to a wide variety of homogeneous, core-fucosylated N-glycopeptides and N-glycoproteins that are hitherto difficult to obtain for structural and functional studies.
Project description:<b>Rationale:</b> Epithelial-mesenchymal transition (EMT) has been recognized as an important step toward high invasion and metastasis of many cancers including hepatocellular carcinoma (HCC), while the mechanism for EMT promotion is still ambiguous. <b>Methods:</b> The dynamic alterations of site-specific glycosylation during HGF/TGF-β1-induced EMT process of three HCC cell lines were systematically investigated using precision glycoproteomic methods. The possible roles of EMT-related glycoproteins and site-specific glycans were further confirmed by various molecular biological approaches. <b>Results:</b> Using mass spectrometry-based glycoproteomic methods, we totally identified 2306 unique intact glycopeptides from SMMC-7721 and HepG2 cell lines, and found that core-fucosylated glycans were accounted for the largest proportion of complex <i>N</i>-glycans. Through quantification analysis of intact glycopeptides, we found that the majority of core-fucosylated intact glycopeptides from folate receptor α (FOLR1) were up-regulated in the three HGF-treated cell lines. Similarly, core-fucosylation of FOLR1 were up-regulated in SMMC-7721 and Hep3B cells with TGF-β1 treatment. Using molecular approaches, we further demonstrated that FUT8 was a driver for HGF/TGF-β1-induced EMT. The silencing of FUT8 reduced core-fucosylation and partially blocked the progress of HGF-induced EMT. Finally, we confirmed that the level of core-fucosylation on FOLR1 especially at the glycosite Asn-201 positively regulated the cellular uptake capacity of folates, and enhanced uptake of folates could promote the EMT of HCC cells. <b>Conclusions:</b> Based on the results, we proposed a potential pathway for HGF or TGF-β1-induced EMT of HCC cells: HGF or TGF-β1 treatment of HCC cells can increase the expression of glycosyltransferase FUT8 to up-regulate the core-fucosylation of <i>N</i>-glycans on glycoproteins including the FOLR1; core-fucosylation on FOLR1 can then enhance the folate uptake capacity to finally promote the EMT progress of HCC cells.