A1 Ions: Peptide-Specific and Intensity-Enhanced Fragment Ions for Accurate and Multiplexed Proteome Quantitation

Accurate proteome quantitation is
of great significance to deeply
understand various cellular and physiological processes. Since a1
ions, generated from dimethyl-labeled peptides, exhibited high formation
efficiency (up to 99%) and enhanced intensities (2.34-fold by average)
in tandem mass spectra, herein, we proposed an a1 ion-based proteome
quantitation (APQ) method, which showed high quantitation accuracy
(relative errors < 7%) and precision (median coefficients of variation
≤ 11%) even in a 20-fold dynamic range. Notably, due to the
mass differences of a1 ions from peptides with different N-terminal
amino acids, APQ demonstrated interference-free capacity by distinguishing
target peptides from the coisolated ones. By designing an isobaric
dimethyl labeling strategy, we achieved simultaneous proteome-wide
measurements across up to eight samples. Using APQ to quantify the
time-resolved proteomic profiles during a TGF-β-induced epithelial-mesenchymal
transition, we found many differentially expressed proteins associated
with fatty acid degradation, indicating that fatty acid metabolism
reprogramming occurred during the process. The APQ method combines
high quantitation accuracy with multiplexing capacity, which is suitable
for deep mining and understanding of dynamic biological processes.