Fig. 1: The MuPPE platform enables integrated multi-level proteomics with enhanced efficiency and coverage.

a Schematic workflow of the MuPPE platform. Protein aggregation capture (PAC), on-bead digestion, and serial enrichment of glycopeptides, phosphopeptides, and proteome are integrated into a single pipeline. Key steps (denaturation, aggregation, digestion, enrichment) and their approximate timeframes are depicted. b The design rationale of MuPPE. Graphical summary of MuPPE’s core design principles, centered on on-bead multi-function integration to address proteomic workflow challenges. Image(s) provided by Servier Medical Art (https://smart.servier.com), licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). c Base peak chromatograms of digested human serum samples prepared with in-solution method (top) and MuPPE (bottom), showing comparable MS pattern profiles. d Comparative identification of precursors, peptides, and protein groups in human serum by in-solution (purple) and MuPPE (blue) methods, demonstrating increased identifications by MuPPE. e Correlation of protein identification coverage between in-solution and MuPPE methods (R² = 0.88), indicating minimal bias in MuPPE. f Coefficient of variation (CV%) distribution of protein quantifications by in-solution (purple) and MuPPE (blue) methods, illustrating higher precision of MuPPE. Each data point in this figure (f) represents the CV% of one individual protein quantified by either the in-solution method (purple) or MuPPE method (blue). Centre line indicates the median; box limits denote the 25th and 75th percentiles; whiskers represent 1.5 × the interquartile range; points show individual quantified features. g Venn diagrams showing overlap of identified glycopeptides/glycoproteins (human serum, left) and phosphopeptides/phosphoproteins (mouse brain, right) by in-solution and MuPPE methods, highlighting expanded coverage by MuPPE. For Figure (c–g), n = 2 technical replicates per method, error bars presented as mean ± SD.