Sequential selective enrichment of phosphopeptides and glycopeptides using amine-functionalized magnetic nanoparticles

As two of the most common and important post-translational modifications (PTMs) of proteins, glycosylation and phosphorylation play critical roles in biological processes. Because of the low abundance of phosphopeptides/glycopeptides, specific and sensitive strategies are especially indispensable for the identification of protein phosphorylation and glycosylation by mass spectrometry (MS). However, most of those previously reported methods only focused on enriching either phosphopeptides or glycopeptides rather than enriching both of them. In the present study, amine-functionalized magnetic nanoparticles were synthesized in a one-pot procedure and successfully used for selective enrichment of both phosphopeptides and glycopeptides. The selectivity of this method was demonstrated by analyzing the mixture of peptides/phosphopeptides/glycopeptides at molar ratio of 10 : 1 : 1; the post-enrichment recovery was 88% and 76% for phosphopeptides and glycopeptides respectively. The sensitivity was at the fmol level for both of the phosphopeptides and glycopeptides. In addition, sequence coverage was increased from 25.6% to 51.8% corresponding to a 102% increase for a model protein asialofetuin. These newly identified phosphopeptides or glycopeptides provided additional sequence information, which was beneficial to the protein identification.