Comprehensive PTM profiling with SCASP-PTM uncovers mechanisms of p62 degradation and ALDOA-mediated tumor progression

Multiple post-translational modification (PTM) proteomics typically combines PTM enrichment with multiplex isobaric labeling and peptide fractionation. However, effective methods for sequentially enriching multiple PTMs from a single sample for data-independent acquisition mass spectrometry (DIA-MS) remain lacking. We present SDS-cyclodextrin-assisted sample preparation (SCASP)-PTM, an approach that enables desalting-free enrichment of diverse PTMs, including phosphopeptides, ubiquitinated peptides, acetylated peptides, glycopeptides, and biotinylated peptides. SCASP-PTM uses SDS for protein denaturation, which is sequestered by cyclodextrins before trypsin digestion, facilitating sequential PTM enrichment without additional purification steps. Combined with DIA-MS, SCASP-PTM quantifies the proteome, ubiquitinome, phosphoproteome, and glycoproteome in HeLa-S3 cell samples, identifying serine 28 phosphorylation as a key driver of poly(I:C)-induced p62 degradation. This method also quantifies PTMs in clinical tissue samples, revealing the critical role of ALDOA K330 ubiquitination/acetylation in tumor progression. SCASP-PTM offers a streamlined workflow for comprehensive PTM analysis in both basic research and clinical applications.