Fig. 2: The feasibility of incorporating glycosylation workflow into MuPPE.

The optimized enzymatic digestion parameters of the MuPPE workflow. a–c Comparison of identified human serum protein groups (a), glycopeptides (b), and the glycopeptides selectivity (c) when digested with E/S ratio of 1 : 50 in digestion time from 1 to 12 h (n = 2). Comparative analysis of the numbers of glycopeptides (d) and the corresponding selectivity of glycopeptides (e) identified using different hydrophilic beads in human serum (n = 3). f The glycosylation identification results of glycopeptides, glycoproteins, and the selectivity of glycopeptides from different operators with mouse brain lysates (n = 2). Oper: Operator. g Sialylated and neutral glycosylation levels comparison across various biological samples (n = 2; MB: mouse brain, HS: human serum, HCSF: human CSF). h The glycosylation identification of HCSF. Gpeps, GPs, and N-glcycosites are the abbreviations for glycopeptides, glycoproteins, and N-glycosylation sites (n = 3). i The N-glycosylation sites of MB, HS, and HCSF identified with MuPPE relative to those reported in UniProt, as summarized in the GlyGen dataset (Version 2.6.1, released on 08/07/2024), and [https://data.glygen.org/GLY_000039] for mouse N-glycosylation sites dataset and [https://data.glygen.org/GLY_000038] for human N-glycosylation sites dataset. Error bars presented as mean ± SD.