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

ABSTRACT: Isobaric chemical tag labeling (e.g., iTRAQ and TMT) is a commonly used approach in quantitative proteomics research. Typically, peptides are covalently labeled with isobaric chemical tags, and quantification is enabled through detection of low-mass reporter ions generated after MS2 fragmentation. Recently, we have introduced and optimized a platform for intact protein-level TMT labeling that demonstrated >90% labeling efficiency in complex sample with top-down proteomics. Higher-energy collisional dissociation (HCD) is a commonly utilized fragmentation method for peptide-level isobaric chemical tag labeling because it produces accurate reporter ion intensities and avoids the loss of low mass ions. HCD energies have been optimized for peptide-level isobaric chemical tag labeling; however, fragmentation energies have not been systematically evaluated for TMT-labeled intact proteins for both protein identification and quantitation. In this study, we report a systematic evaluation of normalized HCD fragmentation energies on TMT-labeled HeLa lysate with top-down proteomics. Our results suggested that reporter ions often require higher collisional energy for higher ion intensities while most of intact proteins fragment when normalized HCD energies are between 30% and 50%. We further demonstrated that a stepped HCD fragmentation scheme with energies between 30 and 50% resulted in the optimized quantitation and identification for TMT-labeled intact HeLa protein lysate by providing average reporter ion intensity as > 3.60 E4 and average PrSM as > 1000 PrSM counts with high confidence.

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yanting Guo  

LAB HEAD: Si Wu

PROVIDER: PXD037491 | Pride | 2023-01-17

REPOSITORIES: Pride