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

Abstract

AbstractTo understand implications of protein glycosylation for clinical diagnostic and biopharmaceuticals, innovative glycoproteomic technologies are required. Recently, significant advances were made, particularly toward structure-focusedN-glyco-proteomic analyses. The mass spectrometric analysis of intactN-glycopeptides using stepped collision fragmentation along with glycan oxonium ion profiling now enables to reliably discriminate between differentN-glycan structures. Still, there are weaknesses that currentN-glycoproteomic approaches must overcome: 1) handling of incorrect identifications, 2) identification of rare and modifiedN-glycans, and 3) insufficient glycoproteomic coverage, especially in complex samples. To address these shortcomings, we have developed an innovativeN-glycoproteomic workflow that aims at providing comprehensive site-specific and structuralN-glycoproteomic data on human blood plasma glycoproteins. The workflow features protein depletion plus various fractionation strategies and the use of high-resolution mass spectrometry with stepped collision fragmentation. Furthermore, by including a decision tree procedure established for data validation, we could significantly improve the description of theN-glycan micro-heterogeneity. Our data analysis workflow allows the reliable differentiation of ambiguousN-glycan structures like antenna-versus core-fucosylation plus the modified and rareN-glycans such as sulfated and glucuronidated ones. With this workflow, we were able to advance in the analysis of human blood plasma glycoproteins to concentrations as low as 10 pg/mL. A total of 1,929N-glycopeptides and 942N-glycosites derived from 805 human middle-to low-abundant glycoproteins were identified. Overall, the presented workflow holds great potential to improve our understanding of protein glycosylation and to foster the discovery of blood plasma biomarkers.Abstract Figure