Achieving\na 35-Plex Tandem Mass Tag Reagent Set through\nDeuterium Incorporation

Mass\nspectrometry-based sample multiplexing with isobaric\ntags\npermits the development of high-throughput and precise quantitative\nbiological assays with proteome-wide coverage and minimal missing\nvalues. Here, we nearly doubled the multiplexing capability of the\nTMTpro reagent set to a 35-plex through the incorporation of one deuterium\nisotope into the reporter group. Substituting deuterium frequently\nresults in suboptimal peak coelution, which can compromise the accuracy\nof reporter ion-based quantification. To counteract the deuterium\neffect on quantitation, we implemented a strategy that necessitated\nthe segregation of nondeuterium and deuterium-containing channels\ninto distinct subplexes during normalization procedures, with reassembly\nthrough a common bridge channel. This multiplexing strategy of “design\nindependent sub-plexes but acquire together” (DISAT) was used\nto compare protein expression differences between human cell lines\nand in a cysteine-profiling (i.e., chemoproteomics) experiment to\nidentify compounds binding to cysteine-113 of Pin1.