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

Area of Science:

• Proteomics
• Molecular Biology
• Biochemistry

Background:

• Investigating multiple protein post-translational modifications (PTMs) is crucial for understanding protein regulation.
• Simultaneous analysis of diverse PTMs is challenging due to limitations in throughput, sensitivity, and reproducibility, especially with limited sample amounts.

Purpose of the Study:

• To develop a robust and efficient workflow for simultaneous analysis of multiple PTMs.
• To address the limitations of existing proteomics workflows for multi-PTM analysis.

Main Methods:

• Developed MoSAIC, a multi-PTM workflow integrating co-enrichment, multiplexing, fractionation, hybrid data acquisition, and unified data analysis.
• Targeted phosphorylation, glycosylation, acetylation, and ubiquitination for simultaneous interrogation.
• Utilized fractionation and tandem mass tag (TMT) labeling for concurrent identification and quantification.

Main Results:

• MoSAIC doubles PTM coverage (4 vs. 2 PTMs) compared to the traditional CPTAC workflow within the same instrument time (24 MS runs).
• Achieved increased identifications of PTM-modified peptides.
• Enhanced throughput and data consistency through concurrent identification and quantification.

Conclusions:

• MoSAIC provides a cost-effective and efficient platform for comprehensive multi-PTM analysis.
• This workflow overcomes key limitations in multi-PTM proteomics, advancing biological and clinical research.