Achieving a 35-Plex 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 doubled the multiplexing capability of the
TMTpro reagent set to a 35-plex through the incorporation of one deuterium
isotope into the reporter group. Substituting deuterium frequently
results in suboptimal peak coelution, which can compromise the accuracy
of reporter ion-based quantification. To counteract the deuterium
effect on quantitation, we implemented a strategy that necessitated
the segregation of nondeuterium and deuterium-containing channels
into distinct subplexes during normalization procedures, with reassembly
through a common bridge channel. This multiplexing strategy of “design
independent sub-plexes but acquire together” (DISAT) was used
to compare protein expression differences between human cell lines
and in a cysteine-profiling (i.e., chemoproteomics) experiment to
identify compounds binding to cysteine-113 of Pin1.