Achieving a 35plex Tandem Mass Tag Reagent Set Through Deuterium Incorporation

Mass spectrometry-based sample multiplexing with isobaric tags permits the development of high-throughput and precise quantitative biological assays with proteome-wide coverage and minimal missing values. Here, we nearly double the multiplexing capability of the TMTpro reagent set to 35-plex through the incorporation of one deuterium isotope into the reporter group. Substituting deuterium frequently results in suboptimal peak co-elution, which can compromise the accuracy of reporter ion-based quantification. To counteract the impact of the deuterium effect on quantitation, we implemented a strategy that necessitated the segregation of non-deuterium and deuterium-containing channels into distinct sub-plexes during normalization procedures with reassembly through a common bridge channel. This multiplexing strategy of “design independent sub-plexes but acquire together” was used to compare protein expression differences between human cell lines and in a cysteine-profiling (e.g., chemoproteomics) experiment to identify compounds binding to cysteine-113 of Pin1.