MoSAIC: An Integrated and Modular Workflow for Confident Analysis of Protein Post-Translational Modification Landscapes

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 when sample amounts are limited. To address these limitations, we present MoSAIC, a multi-PTM workflow integrating coenrichment 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 interrogation of these modifications simultaneously. Compared with the traditional Clinical Proteomic Tumor Analysis Consortium workflow, MoSAIC doubles PTM coverage (four versus two PTMs) while maintaining the same instrument time (24 mass spectrometry runs), achieving increased identifications of PTM-modified peptides. By leveraging fractionation and tandem mass tag 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.