N-terminal blocking sialic acid linkage specific alkylamidation for qualitative and quantitative analysis of sialytated glycoprotein
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Zhixin Tian
PROVIDER: PXD072001 | iProX | Mon Dec 15 00:00:00 GMT 2025
REPOSITORIES: iProX
Similar Datasets
Project description:In this study, we provide a simple and straightforward method to distinguish core-fucosylated glycans from antenna-fucosylated glycans by systematically evaluating the extent of fucose migration based on the feature Y ions by using Fut8 knockout (Fut8−/−) mouse brain under HCD energy of 20%. The method was further evaluated by multiple different approaches including validation by standard glycopeptides, evaluation in silico, comparison under the lower energy (15%), and applied to analyze core-fucosylated glycans in wide-type (Fut8+/+) mouse brain.
2022-11-14 | PXD035158 | Pride
Project description:Background Plasma proteins can be modified by addition of sugar residues by non-enzymatic glycation as well as glycosylation. While glycation is a hallmark of hyperglycemia, glycosylation is a complex, enzyme-catalyzed post-translational modification by heterogeneous sugar chains and is present on a majority of plasma proteins. N-linked glycosylation occurs on asparagine residues occurring predominantly on a canonical N-glycosylation motif (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported to be glycosylated less frequently. Albumin is the most abundant protein in human plasma whose glycation has been implicated in end-organ damage in advanced diabetes mellitus, and as such, has both diagnostic and therapeutic implications. However, albumin has long been regarded as a non-glycosylated protein owing to the absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated with two possible attached glycans. Methods To investigate N-linked glycosylation of abundant serum proteins in greater detail, we passed healthy donor samples through a multiple affinity removal spin (MARS14) column followed by trypsin digestion of bound proteins and subsequent glycopeptide enrichment by size-exclusion chromatography (SEC) or mixed-mode anion-exchange (MAX), in parallel. Global analysis of intact glycopeptides was performed by LC-MS/MS to evaluate potential glycosylation at canonical as well as non-canonical sites. PNGase F treatment was carried out to confirm N-linked glycosylation at non-canonical sites Asn-X-Cys/Val. Glycopeptides on albumin at non-canonical sites were further characterized by MS3 fragmentation from immunoprecipitated albumin to confirm the attached glycans. To assess if the observed glycosylation was a general phenomenon, targeted analysis using parallel reaction monitoring (PRM) was carried out on an independent set of twenty human serum samples. Finally, to test whether albumin glycosylation is unique to human albumin or is a general phenomenon, bovine and rabbit serum albumins were subjected to MAX chromatography and LC-MS/MS analysis. Results We report that human albumin is modified by N-linked glycosylation at two sites, Asn68(-Glu-Val) and Asn123-(Glu-Cys), both of which occur in non-canonical N-glycosylation motifs. We detected N-glycopeptides at both sites bearing four complex mono- and bi-antennary N-linked glycans, with Hex5HexNAc4NeuAc2 and Hex5HexNAc4NeuAc1 being the most abundant glycans. These findings were validated by analyzing deglycosylated peptides and MS3-based fragmentation of glycopeptides. Glycosylation at both sites was confirmed by targeted MS in twenty donor samples. Finally, we report that the Asn residue on bovine and rabbit serum albumins that corresponds to the highly conserved Asn123 in human albumin is also N-glycosylated with complex sialylated glycans similar to those observed in human albumin. Conclusions Albumin is a conserved glycoprotein with multiple N-linked glycosylation sites. This glycosylation of albumin has potential functional implications and applications in medicine.
2024-06-16 | PXD047863 | Pride
Project description:In eukaryotic cells, unconjugated oligosaccharides structurally related to N-glycans (FNGs) are generated either from misfolded N-glycoproteins destined for endoplasmic reticulum (ER)-associated degradation (ERAD), or from lipid-linked oligosaccharides, donor substrates for N-glycosylation of proteins. The mechanism responsible for the generation of FNGs is now well understood, but whether there are other type of free glycans remain unclarified. Here, we report on the accumulation of O-mannosylated free glycans in budding yeast that were cultured in media containing mannose as a carbon source. A structural analysis of these glycans revealed that their structures are identical to those of O-mannosyl glycans that are attached to glycoproteins. Deletion of the cyc8 gene, encoding a general transcription repressor, resulted in the accumulation of excessive amounts of the free O-glycans, concomitant with a severe growth defect, a reduction in the level of cellular O-mannosylated proteins, and compromised cell wall integrity. Our findings provide evidence for a regulated pathway for O-glycoprotein degradation in yeast and offer critical insights into the catabolic mechanisms that control the fate of O-glycosylated proteins.
2019-07-24 | GSE130332 | GEO
Project description:Protein core-fucosylation plays a crucial role in regulating cell surface protein functions and is involved in various biological processes, including cell signaling, immune response, and cancer progression. However, core-fucosylation (CF) poses particular challenges in identification and characterization due to its low abundance and the high heterogeneity of N-glycosylation. To overcome these challenges, this study systematically investigated the oxidation mechanisms of core-fucose and developed a selective enrichment strategy that combines chemical oxidation with glycan truncation for cell surface core-fucosylation characterization. Specifically, sodium periodate was employed to selectively oxidize cell surface glycans. In combination with the endoglycosidases Endo M and Endo F3, which possessed complementary substrate specificity and broad tolerance, this approach efficiently truncated N-glycans and leaving core-fucosylated glycopeptides bearing aldehyde tags. Utilizing reversible hydrazide chemistry, core-fucosylated glycopeptides were selectively enriched. The developed strategy was applied to profile cell surface core-fucosylatd protein in HeLa cells, which yielded 74 core-fucosylated glycopeptides corresponding to 21 key cell surface drug targets, thereby validating the efficacy of this approach. Collectively, this study systematically investigatd the mechnism of core-fucosylation oxidation and developed a new technical tool for studying cell surface protein core-fucosylation.
2025-12-09 | PXD064328 | JPOST Repository
Project description:The dense O-glycosylation of mucins plays an important role in the defensive properties of the mucus hydrogel. Aberrant glycosylation is often correlated with inflammation and pathology such as COPD, cancer, and Crohn’s disease. The inherent complexity of glycans and the diversity in the O-core structure constitute fundamental challenges for the analysis of mucin-type O-glycans. Due to coexistence of multiple isomers, multidimensional workflows such as LC-MS are required. To separate the highly polar carbohydrates, porous graphitized carbon is often used as a stationary phase. However, LC-MS workflows are time-consuming and lack reproducibility. Here we present a rapid alternative for separating and identifying O-glycans released from mucins based on trapped ion mobility mass spectrometry. Compared to established LC-MS, the acquisition time is reduced from an hour to two minutes. To test the validity, the developed workflow was applied to sputum samples from cystic fibrosis patients to map O-glycosylation features associated with disease.
2024-03-19 | PXD050530 | Pride
Project description:Background:Gastric cancer (GC) is one of the most aggressive gastrointestinal malignancy and leading cause of cancer related deaths worldwide,warranting the urgent discovery of biomarkers for early detection. IgG glycosylation is strongly related to disease progression and specific IgG glycans have been regarded as the next-generation biomarker, whereas it is rarly studied on the association between subclass-specific IgG glycosylation profile and GC progression. Methods:A total of 141 individuals were enrolled and randomly sorted into discovery set consisting of normal (N=30) and cancer patients (N=20), and validation set including controls (N=30), benign (N=25) and cancer (N=36).IgG glycopeptides were tryptic digested and enrichmed by microcrystalline cellulose based solid phase extraction.Subclass-specific IgG N-glycosylation was analyzed by nanoLC-MS/MS. Results:It was found that GC progression was associated with increased sialylation and decreased bisection of IgG2, as well as substantially decreased sialylation of IgG1 and increased galactosylation of IgG3/4.Further analysis of individual glycopeptides showed the changes of particular glycosylation of IgGwere reflected by the specific glycans taht could apparently distinguish different stages of gastric disease patients from normal. Conclusions:Different subclasses of IgG glycosylation were differentially expressed in GC, specific IgG glycopeptides could be promising biomarker for early detection of GC patients.
2025-05-06 | PXD027475 | Pride
Project description:The inter-alpha-trypsin inhibitor (IαI) complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The IαI complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcA3Gal3Gal4Xyl-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the non-sulfated non-reducing end, (GalNAc4GlcA3)n ,to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation (HCD). The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal two GalNAc residues. The fragmentation behavior of CS glycopeptides under variable HCD conditions displays an energy dependency that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.
2015-10-02 | PXD002707 | Pride
Project description:<h4><strong>BACKGROUND:</strong> Multiple myeloma is characterized by clonal proliferation of malignant plasma cells in the bone marrow that produce monoclonal immunoglobulins. N-glycosylation changes of these monoclonal immunoglobulins have been reported in multiple myeloma, but previous studies only detected limited serum N-glycan features.</h4><h4><strong>METHODS:</strong> Here, a more detailed study of the human serum N-glycome of 91 multiple myeloma patients and 51 controls was performed. We additionally analyzed sequential samples from patients (n = 7) which were obtained at different time points during disease development as well as 16 paired blood serum and bone marrow plasma samples. N-glycans were enzymatically released and measured by mass spectrometry after linkage specific derivatization of sialic acids.</h4><h4><strong>RESULTS:</strong> A decrease in both α2,3- and α2,6-sialylation, galactosylation and an increase in fucosylation within complex-type N-glycans were found in multiple myeloma patients compared to controls, as well as a decrease in difucosylation of diantennary glycans. The observed glycosylation changes were present in all ISS stages, including the 'low-risk' ISS I. In individual patients, difucosylation of diantennary glycans decreased with development of the disease. Protein N-glycosylation features from blood and bone marrow showed strong correlation. Moreover, associations of monoclonal immunoglobulin (M-protein) and albumin levels with glycan traits were discovered in multiple myeloma patients.</h4><h4><strong>CONCLUSIONS & GENERAL SIGNIFICANCE: </strong>In conclusion, serum protein N-glycosylation analysis could successfully distinguish multiple myeloma from healthy controls. Further studies are needed to assess the potential roles of glycan trait changes and the associations of glycans with clinical parameters in multiple myeloma early detection and prognosis.</h4>
2022-04-27 | MTBLS676 | MetaboLights
Project description:We employed the newly available IMPa O-glycoprotease from Pseudomonas aeruginosa for O-glycoproteomics analysis of cultured cells and tissues. The glycopeptides were extracted, purified, and conjugated to a solid support before an enzymatic cleavage by IMPa. O-glycopeptides were analyzed by EThcD, which allowed for a site-specific and global analysis of O-glycans, especially those sialylated and the Tn antigen. Through wild-type or SimpleCell HEK293 cells, we present two approaches, one for the analysis of total O-glycoproteome and the other for the Tn glycoproteome, and showed that IMPa and EThcD are necessary for confident localization of O-glycans on glycopeptides. We applied this method to study the O-glycoproteome of mouse brain, which revealed the high frequency of sialylated O-glycans and the Tn antigen on brain glycoproteins.
2023-08-28 | PXD037415 | Pride
Project description:The spondylodysplastic type of Ehlers-Danlos syndromes (spEDS) is caused by genetic defects in the B4GALT7 or B3GALT6 genes both deranging the biosynthesis of the glycosaminoglycan linkage region of chondroitin/dermatan sulfate and heparan sulfate proteoglycans. In this study we have analyzed the linkage regions of urinary chondroitin sulfate proteoglycans of three siblings, diagnosed with spEDS and carrying biallelic pathogenic variants of the B3GALT6 gene. Proteoglycans were digested with trypsin, glycopeptides enriched on anion-exchange columns, depolymerized with chondroitinase ABC and analyzed by nLC-MS/MS. In urine of the unaffected mother, the dominating glycopeptide of bikunin/protein AMBP appeared as only one dominating (99.9%) peak with the canonical tetrasaccharide linkage region modification. In contrast, the samples of the three affected siblings contained two different glycopeptide peaks, corresponding to the canonical tetrasaccharide and to the non-canonical trisaccharide linkage region modifications in individual ratios of 61/38, 73/27, 59/41.We propose that the relative distribution of glycosaminoglycan linkage regions of urinary bikunin glycopeptides may serve as a phenotypic biomarker in a diagnostic test but also as a biomarker to follow the effect of future therapies in affected individuals.
2022-06-29 | PXD033230 | Pride