Integrated Analysis of Posttranslational Modifications and Total Proteome: Methods for Distinguishing Abundance from...

Posttranslational modifications (PTMs), particularly phosphorylation, regulate protein function at substoichiometric levels, making their quantitative analysis technically challenging. A critical limitation in PTM studies is distinguishing between changes in modification abundance due to altered protein expression versus genuine changes in modification stoichiometry (the fraction of protein molecules that are modified). This chapter presents a comprehensive workflow integrating Tandem-Mass-Tag (TMT)-based quantitative proteomics with specialized computational analysis to distinguish differential PTM abundance (DPA) from differential PTM usage (DPU) using the prolfqua, prolfquapp, and prophosqua R packages. The workflow introduces a robust statistical framework for integrated PTM-protein analysis; comprehensive visualization tools, including N-terminus to C-terminus (N-to-C) plots for protein-centric PTM mapping and sequence motif analysis for kinase prediction. This integrated approach enables researchers to identify actual signaling changes in PTM studies, prioritize functionally relevant modification sites, understand pathway-level regulation in disease contexts, and generate robust datasets suitable for publication in high-impact journals.