Systems analysis of singly and multiply O-glycosylated peptides in the human serum glycoproteome via EThcD and HCD mass spectrometry

Abstract Human serum has been intensively studied to identify biomarkers via global proteomic analysis. The altered O -glycoproteome is associated with human pathological state including cancer, inflammatory and degenerative diseases and is an attractive source of disease biomarkers. Because of the microheterogeneity and macroheterogeneity of O -glycosylation, site-specific O -glycosylation analysis in human serum is still challenging. Here, we developed a systematic strategy that combined multiple enzyme digestion, multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O -glycopeptide characterization. We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD is not only suitable for the identification of singly O -glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O -glycosylated peptides. Totally, with the strict scoring criteria, 499 non-redundant intact O -glycopeptides, 173 O -glycosylation sites and 6 types of O -glycans originating from 49 O -glycoprotein groups were identified in human serum, including 121 novel O -glycosylation sites. Currently, this is the largest data set of site-specific native O -glycoproteome from human serum samples. We expect that the strategies developed by this study will facilitate in-depth analyses of native O -glycoproteomes in human serum and provide opportunities to understand the functional roles of protein O -glycosylation in human health and diseases. Biological significance The altered O -glycoproteome is associated with human pathological state and is an attractive source of disease biomarkers. However, site-specific O -glycosylation analysis is challenging because of the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of O -glycosylation. In this work, we developed a systematic strategy for intact O -glycopeptide characterization. This study took advantage of the inherent properties of the new fragmentation method called EThcD, which provides more complete fragmentation information about O -glycosylated peptides and a more confident site localization of O -glycans than collision-induced dissociation (HCD). We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD was not only suitable for the identification of singly O -glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O -glycosylated peptides. Finally, we got a largest data set of site-specific native O -glycoproteome from human serum samples. Furthermore, quantitative analysis of intact O -glycopeptides from the serum samples of IgA nephropathy (IgAN) patients and healthy donors was performed, and the results showed the potential of the strategy to discover O -glycosylation biomarkers. We expect that the strategies developed by this study will facilitate in-depth analyses of native O -glycoproteomes in human serum and lead to exciting opportunities to understand the functional roles of protein O -glycosylation in human health and diseases.