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.