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.

Sex-hormone-binding globulin (SHBG) is a liver-secreted glycoprotein and a major regulator of steroid distribution. It has been reported that the serum concentration of SHBG changes in liver disease. To explore the involvement of SHBG in liver disease of different etiologies in greater detail, we developed a sensitive and selective liquid chromatography-mass spectrometry parallel reaction monitoring workflow to achieve quantitative analysis of SHBG glycosylation microheterogeneity. The method uses energy-optimized "soft" fragmentation to extract informative Y ions for maximal coverage of glycoforms and their quantitative comparisons. A total of 15 N-glycoforms of two N-glycosites and 3 O-glycoforms of 1 O-glycosite of this low-abundance serum protein were simultaneously analyzed in the complex samples. At the same time, we were able to partially resolve linkage isoforms of the fucosylated glycoforms and to identify and quantify SHBG N-glycoforms that were not previously reported. The results show that both core and outer-arm fucosylation of the N-glycoforms increases with liver cirrhosis but that a further increase of fucosylation is not observed with hepatocellular carcinoma (HCC). In contrast, the α-2-6 sialylated glycoform of the O-glycopeptide of SHBG increases in liver cirrhosis, and a significant 2-fold further increase is observed in HCC. In general, we do not find a significant contribution of different liver disease etiologies to the observed changes in glycosylation; however, elevation of the newly reported HexNAc(4)Hex(6) N-glycoform is associated with alcoholic liver disease.

Introduction: N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membraneassociated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy.Methods: Mass spectrometry was used to analyze the distribution of the sitespecific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment.Results: While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane.Conclusions: Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.

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 Nglycopeptides. 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.