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
Publisher
Cold Spring Harbor Laboratory
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