Extensive heterogeneity of glycopeptides in plasma revealed by deep glycoproteomic analysis

ABSTRACT: A number of plasma glycoproteins are clinically useful as biomarkers in a variety of diseases. Although thousands of proteins are present in plasma, >95% of the plasma proteome by mass is represented by only 22 proteins. This necessitates strategies to deplete the abundant proteins and enrich other subsets of proteins. Although glycoproteins are abundant in plasma, in routine proteomic analyses, glycopeptides are not often investigated. Traditional methods such as lectin-based enrichment of glycopeptides followed by deglycosylation have helped understand the glycoproteome, but they lack any information about the attached glycans. Here, we apply size-exclusion chromatography (SEC) as a simple strategy to enrich intact glycopeptides based on their larger size which achieves broad selectivity regardless of the nature of attached glycans. Using this approach, we identified 1,317 glycopeptides belonging to 266 glycosylation sites on 154 plasma glycoproteins. The deep coverage achieved by this approach was evidenced by extensive heterogeneity that was observed. For instance, 20-100 glycopeptides were observed per protein for the top 15 glycoproteins. Overall, we found 615 novel glycopeptides of which 39 glycosylation sites (from 38 glycoproteins) are not including in protein databases such as Uniprot and GlyconnectDB. We also identified 12 novel glycopeptides containing di-sialic acid, which is a rare glycan epitope. Thus, SEC allows for efficient LC-MS/MS-based deep glycoproteomics from low amounts (~1 l) of human plasma. Overall, the SEC-based method described here is a simple, rapid and high-throughput strategy for characterization of the glycoproteome.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Akhilesh Pandey  

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD025414 | Pride | 2021-08-18

REPOSITORIES: Pride