A Tribrid Hyperplexing approach for Large-scale Quantitative Proteomic Analysis

Isobaric labeling has emerged as an indispensable quantitative proteomic approach for its unprecedented multiplexing capacity in a single analysis. Currently, different hyperplexing approaches have been developed to meet the need for increasing sample numbers in large-scale cohort analysis. In this report, we present a tribrid hyperplexing approach by the combinational use of three types of isobaric reagents, a novel isobaric tag 16-plex (IBT16) reagent, and the widely used TMT (TMT11) and TMTpro (TMT18) reagents. After the determination of the labeling efficiency and the optimization testing conditions, we systematically evaluated the identification and quantification performance of the three labeling methods in both independent and combinatorial means using the mixtures of E. coli and HeLa peptides with different ratios. Our results reveal that the three reagents are quite similar in all testing aspects although with some differences, and the combination use of three reagents to expand the multiplexing capacity is feasible. Furthermore, we provide practical recommendations for the choice of isobaric reagent combinations according to different plexes.