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

## TL;DR

MoSAIC is a new workflow for analyzing multiple protein modifications at once, improving efficiency and accuracy in proteomics research.

## Contribution

MoSAIC introduces a modular workflow enabling simultaneous analysis of four PTMs with improved throughput and reproducibility.

## Key findings

- MoSAIC doubles PTM coverage compared to existing workflows while maintaining instrument time.
- The workflow quantified over 50,000 PTM-modified peptides in breast cancer xenografts.
- Hybrid DDA/DIA acquisition reduces platform-specific bias and improves data consistency.

## Abstract

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.

•Introduced acetylated/ubiquitinated peptide coenrichment to streamline workflow.•Postenrichment TMT multiplexing enables DIA and preserves Gly-Gly antibody recognition.•Pooled four PTM enrichments into one TMT mix, cutting redundant MS runs.•Hybrid DDA/DIA with cross-validation reduces platform-specific bias.

Introduced acetylated/ubiquitinated peptide coenrichment to streamline workflow.

Postenrichment TMT multiplexing enables DIA and preserves Gly-Gly antibody recognition.

Pooled four PTM enrichments into one TMT mix, cutting redundant MS runs.

Hybrid DDA/DIA with cross-validation reduces platform-specific bias.

Post-translational modifications (PTMs) critically regulate protein function, yet simultaneous analysis of multiple PTMs remains technically challenging. We introduce MoSAIC, a modular and integrated workflow that combines targeted enrichment chemistries, multiplexed tandem mass tag labeling, and hybrid data-dependent acquisition/data-independent acquisition mass spectrometry for robust multi-PTM quantification. MoSAIC enables parallel profiling of phosphorylation, glycosylation, acetylation, and ubiquitination from the same biological sample, while reducing instrument time and improving reproducibility over existing approaches. Applied to breast cancer patient-derived xenografts, the workflow quantified >50,000 PTM-modified peptides and uncovered extensive PTM crosstalk. MoSAIC provides a scalable workflow for multidimensional PTM mapping with broad applications in translational research and biomarker discovery.