Optimization of Higher-Energy Collisional Dissociation Fragmentation Energy for Intact Protein-Level Tandem Mass Tag Labeling

• DOI: 10.1021/acs.jproteome.2c00549
• Publication Date: 2023-01-05T21:57:50Z
• Authors: Yanting Guo; Trishika Chowdhury; Meena Seshadri; Kellye A. Cupp-Su...; Qingyu Wang; Dahang Yu; Si Wu
• Keywords: Fragmentation; Tandem mass tag; Isobaric labeling; Isobaric process; Collision-induced dissociation

Isobaric chemical tag labeling (e.g., TMT) is a commonly used approach in quantitative proteomics, and quantification enabled through detection of low-mass reporter ions generated after MS2 fragmentation. Recently, we have introduced optimized an intact protein-level TMT platform that demonstrated >90% efficiency complex samples with top-down proteomics. Higher-energy collisional dissociation (HCD) utilized for isobaric tag-labeled peptide fragmentation because it produces accurate ion intensities avoids loss low mass ions. HCD energies been labeled-peptides but not systematically evaluated proteins. In this study, report systematic evaluation normalized (NCEs) on TMT-labeled HeLa cell lysate using Our results suggested often result higher at NCEs. Optimal proteins identification, however, required relatively lower NCE. We further stepped NCE scheme from 30% to 50% resulted optimal identification These parameters average intensity ∼4E4 proteoform spectrum matches (PrSMs) >1000 per RPLC-MS/MS run 1% false discovery rate (FDR) cutoff.