A Streamlined Sequential Enrichment Strategy for Multi-PTM Profiling from Low Micrograms of Samples

Post-translational modifications (PTMs) are critical regulators of protein function and are involved in many human diseases. However, comprehensive analysis of multiple PTM types from limited biological material remains a significant challenge, primarily due to the low stoichiometry and sample loss during intricate preparation procedures. Herein, we developed a novel, streamlined sequential enrichment strategy for the simultaneous analysis of the N -glycoproteome, phosphoproteome, ubiquitinome, and global proteome from a single, microscale sample (multi-PTM profiling strategy). Our strategy integrates HILIC beads for N -glycopeptides enrichment, Ti 4+ -IMAC beads for phosphopeptides enrichment, K-ε-GG antibody for ubiquitinated peptides enrichment, and C 18 membrane for global proteome analysis. This entire process was optimized for reducing sample loss, simplifying operation procedures, and reducing time expenditure. Applying this strategy to a single, 20 μg sample of unstimulated and unfractionated human embryonic kidney 293T tryptic peptides, we identified 6229 N -glycosites, 20,726 confident phosphosites, 4368 confident K-ε-GG sites, and 7968 protein groups. Remarkably, even when the peptide input was reduced to 1 μg, 1899 N -glycosites, 11,924 confident phosphosites, 304 confident K-ε-GG sites, and 7268 protein groups were identified. Our strategy was also successfully applied for multi-PTM profiling of complex biological matrices, including 20 μg of mouse tissue peptides and as little as 1 μL of human plasma. We expect that this sensitive and efficient multi-PTM profiling strategy is promising for the PTM crosstalk study and biomarker discovery with precious clinical materials.