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New avenues for human blood plasma biomarker discovery via improved in-depth analysis of the low-abundant N-glycoproteome

ABSTRACT: An in-depth N-glycoproteomic analysis of human blood plasma was achieved by the combination of a top 14-High Abundant Protein (HAP) depletion step and protein fractionation via a GLEFrEE system. The sample preparation workflow included a tryptic digestion and glycopeptide enrichment, followed by an intact N-glycopeptide LC-MS/MS-based analysis, established by us. Two MS/MS acquisitions were applied using different fragmentation energies HCD.step and HCD.low (HCD: Higher Energy Collision Dissociation). A proteomic analysis was conducted, to assess the gain in the identification of middle- and low-abundant proteins. The protein search was applied on the three stages of the workflow: 1) untreated blood plasma, 2) top 14-HAP depleted sample, and 3) top 14-HAP depleted and GELFrEE fractionated sample. (Search engine Proteome Discoverer v 2.5) An N-glycoproteomic analysis was performed on the HCD.low and HCD.step raw data from the top 14-HAP depleted and GELFrEE fractionated sample (search engine Byonic v4.2.10). No FDR cuts nor decoys search was applied and manual validation was conducted instead. After the validation, we achieved the identification of 1929 N-glycopeptides, 942 N-glycosylation sites, and 805 glycoproteins.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Udo Reichl Marcus Hoffmann Erdmann Rapp Frania Jaqueline Zuniga-Banuelos

PROVIDER: MSV000091870 | MassIVE | Sat May 06 10:03:00 BST 2023

SECONDARY ACCESSION(S): PXD042039

REPOSITORIES: MassIVE

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