Comprehensive PTM profiling with SCASP-PTM uncovers mechanisms of p62 degradation and ALDOA-mediated tumor progression
Zhan-Peng Lin, Guohong Gan, Xu Xiao, Chengwen Wen, Xin Ding, Xiangyu Chen, Kaijie Zhang, Wenyu Guo, Mingxin Lin, Yuyang Wang, Xi Chen, Changchuan Xie, Jinling Wang, Minjie Li, Chuan‐Qi Zhong
Abstract
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.