An Integrated Workflow for Analysis of Multiple Protein Post-Translational Modifications

Investigating multiple protein post-translational modifications (PTMs) is critical for unraveling the complexities of protein regulation and the dynamic interplay among PTMs, a growing focus in proteomics. However, simultaneous analysis of diverse PTMs remains a significant technical challenge, as existing workflows struggle to balance throughput, sensitivity, and reproducibility, particularly with limited amount of samples. To address these limitations, we present the first multi-PTM workflow termed MoSAIC, integrating co-enrichment strategies, multiplexing, fractionation, hybrid data acquisition, and unified data analysis, optimized for clinically relevant biological samples. This approach targets phosphorylation, glycosylation, acetylation, and ubiquitination, enabling comprehensive and simultaneous interrogation of these modifications. Compared to conventional methodologies, our strategy reduced total instrument time while identifying more PTM-specific peptides. By leveraging fractionation and tandem mass tag (TMT) labeling, we achieved concurrent identification and quantification of PTM-specific peptides from the same sample, enhancing throughput and data consistency. This robust workflow addresses key limitations in multi-PTM proteomics, providing a cost-effective and efficient platform to advance biological and clinical research.