Analysis of isobaric quantitative proteomic data using TMT-Integrator and FragPipe computational platform

Abstract

Abstract Isobaric mass tags, such as isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tag (TMT), are widely utilized for peptide and protein quantification in multiplex quantitative proteomics. We present TMT-Integrator, a bioinformatics tool for processing quantitation results from TMT and iTRAQ experiments, offering integrative reports at the gene, protein, peptide, and post-translational modification site levels. We demonstrate the versatility of TMT-Integrator using five publicly available TMT datasets: clear cell renal cell carcinoma (ccRCC) whole proteome and phosphoproteome datasets from the Clinical Proteomic Tumor Analysis Consortium, an E. coli dataset with 13 spike-in proteins, and two human cell lysate datasets showcasing the latest advances with the Thermo Orbitrap Astral mass spectrometer and TMTpro 35-plex reagents. Integrated into the widely used FragPipe computational platform ( https://fragpipe.nesvilab.org/ ), TMT-Integrator is a core component of TMT and iTRAQ data analysis workflows. We evaluated the performance of FragPipe coupled with TMT-Integrator analysis pipeline against MaxQuant and Proteome Discoverer with multiple benchmarks, facilitated by the bioinformatics tool OmicsEV. Our results show that FragPipe coupled with TMT-Integrator quantifies more proteins in the E. coli and ccRCC whole proteome datasets, quantifies more phosphorylated sites in the ccRCC phosphoproteome dataset, and overall delivers more robust quantification performance compared to other tools.