Dataset Information

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins.

ABSTRACT: Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270.

SUBMITTER: Hoffmann M

PROVIDER: S-EPMC4739677 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins.

Hoffmann Marcus M Marx Kristina K Reichl Udo U Wuhrer Manfred M Rapp Erdmann E

Molecular & cellular proteomics : MCP 20151123 2

Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-s ...[more]

PMID: 26598643

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Project description:Glycosylation is an important protein modification, which influences the physical and chemical properties as well as biological function of these proteins. Large-scale population studies have shown that the levels of various plasma protein N-glycans are associated with many multifactorial human diseases. Observed associations between protein glycosylation levels and human diseases have led to the conclusion that N-glycans can be considered a potential source of biomarkers and therapeutic targets. Although biochemical pathways of glycosylation are well studied, the understanding of the mechanisms underlying general and tissue-specific regulation of these biochemical reactions in vivo is limited. This complicates both the interpretation of the observed associations between protein glycosylation levels and human diseases, and the development of glycan-based biomarkers and therapeutics. By the beginning of the 2010s, high-throughput methods of N-glycome profiling had become available, allowing research into the genetic control of N-glycosylation using quantitative genetics methods, including genome-wide association studies (GWAS). Application of these methods has made it possible to find previously unknown regulators of N-glycosylation and expanded the understanding of the role of N-glycans in the control of multifactorial diseases and human complex traits. The present review considers the current knowledge of the genetic control of variability in the levels of N-glycosylation of plasma proteins in human populations. It briefly describes the most popular physical-chemical methods of N-glycome profiling and the databases that contain genes involved in the biosynthesis of N-glycans. It also reviews the results of studies of environmental and genetic factors contributing to the variability of N-glycans as well as the mapping results of the genomic loci of N-glycans by GWAS. The results of functional in vitro and in silico studies are described. The review summarizes the current progress in human glycogenomics and suggests possible directions for further research.

Dataset Information

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins.

Publications

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins.

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