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Optimization of N-glycopeptides analysis methods and their preliminary application to barley proteins study
Benkovská, Dagmar ; Flodrová, Dana ; Bobálová, Janette ; Laštovičková, Markéta
N-glycosylation is the most frequently studied plant protein post-translational modification. The analysis of Nglycopeptides after protein proteolytic digestion offers information about the structure of both oligosaccharide and peptide moiety. However, this method has so far been less commonly used. Since glycopeptides hardly ionize during MS analysis in the presence of non-glycosylated peptides, they need to be separated from the complex peptide mixture. In this study, the glycopeptides enrichment, purification and analysis methods were successfully optimized on two standard N-glycoproteins. Concanavalin A (ConA) lectin tips were used for glycopeptide capturing, and obtained fractions were purified on carbon tips and analyzed using MALDI-TOF mass spectrometry. The differences in the CID fragmentation of certain types of glycopeptides were found. This technique was then applied to glycopeptide analysis of barley grain and malt proteins. Several barley glycopeptides were found, however, their identification was very difficult. More proteins separation techniques will be required before this enrichment procedure in further studies.
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