TAG-TMTpro, a Hyperplexing Quantitative Approach for High-Throughput Proteomic Studies.

Isobaric labeling is the most widely used multiplexing quantitative approach in proteomic studies, enabling the comparison of up to 18 samples in a single MS analysis. Expanding the multiplexing capacity is of great necessity for high-throughput proteomic studies. Herein, we establish a novel TAG-TMTpro approach by introducing Ala or Gly residues to peptides prior to TMTpro labeling, which is able to triple the quantitative capacity of TMTpro. We systematically evaluated the Boc-Ala-OSu and Boc-Gly-OSu reaction and optimized the conditions for labeling, side-product elimination, and Boc deprotection. We validated the identification and quantification performance using E. coli and HeLa cell lysates. We demonstrated that the TAG-TMTpro approach resulted in good identification reproducibility and reliable quantitative accuracy. The TAG-TMTpro is able to triple the multiplexing capacity of TMTpro reagents and is a versatile quantitative approach for high-throughput proteomic studies. Data are available via ProteomeXchange with identifier PXD033711.