Dataset Information

Site-Specific Profiling of Serum Glycoproteins Using N-Linked Glycan and Glycosite Analysis Revealing Atypical N-Glycosylation Sites on Albumin and ?-1B-Glycoprotein.

ABSTRACT: Most serum proteins are N-linked glycosylated, and therefore the glycoproteomic profiling of serum is essential for characterization of serum proteins. In this study, we profiled serum N-glycoproteome by our recently developed N-glycoproteomic method using solid-phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) coupled with LC-MS/MS and site-specific glycosylation analysis using GPQuest software. Our data indicated that half of identified N-glycosites were modified by at least two glycans, with a majority of them being sialylated. Specifically, 3/4 of glycosites were modified by biantennary N-glycans and 1/3 of glycosites were modified by triantennary sialylated N-glycans. In addition, two novel atypical glycosites (with N-X-V motif) were identified and validated from albumin and ?-1B-glycoprotein. The widespread presence of these two glycosites among individuals was further confirmed by individual serum analyses.

SUBMITTER: Sun S

PROVIDER: S-EPMC6467210 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Site-Specific Profiling of Serum Glycoproteins Using N-Linked Glycan and Glycosite Analysis Revealing Atypical N-Glycosylation Sites on Albumin and α-1B-Glycoprotein.

Sun Shisheng S Hu Yingwei Y Jia Li L Eshghi Shadi Toghi ST Liu Yang Y Shah Punit P Zhang Hui H

Analytical chemistry 20180501 10

Most serum proteins are N-linked glycosylated, and therefore the glycoproteomic profiling of serum is essential for characterization of serum proteins. In this study, we profiled serum N-glycoproteome by our recently developed N-glycoproteomic method using solid-phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) coupled with LC-MS/MS and site-specific glycosylation analysis using GPQuest software. Our data indicated that half of identified N-glycosites were modified by ...[more]

PMID: 29671580

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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.

Dataset Information

Site-Specific Profiling of Serum Glycoproteins Using N-Linked Glycan and Glycosite Analysis Revealing Atypical N-Glycosylation Sites on Albumin and ?-1B-Glycoprotein.

Publications

Site-Specific Profiling of Serum Glycoproteins Using N-Linked Glycan and Glycosite Analysis Revealing Atypical N-Glycosylation Sites on Albumin and α-1B-Glycoprotein.

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