Super Heavy TMTpro Labeling Reagent: An Alternative and Higher-Charge-State-Amenable Stable-Isotope-Labeled TMTpro Variant.

Stable isotope labeling is a leading strategy for mass-spectrometry-based peptide quantification. Whereas TMTpro isobaric tagging can quantify up to 16 multiplexed samples in a single experiment, nonisobaric, yet chromatographically indistinguishable, variants of TMTpro reagents can be used in conjunction with the isobaric tag series for various peptide-targeting applications. Here we test the performance of two nonisobaric TMTpro variants, a stable-isotope-free TMTproZero tag and a nearly fully isotope-labeled "super-heavy" variant, shTMTpro, in a targeted assay for peptides of charge state 4+. We label each peptide with TMTproZero or Super Heavy TMTpro reagents and separately spike each peptide into a TMTpro16-labeled background (equal amount of peptide across all 16 channels). We observe that the expected 1:1 reporter ion ratio is distorted when a TMTproZero-labeled peptide is used; however, we note no such interference when shTMTpro substitutes the TMTproZero tag. Our data suggest that using the Super Heavy TMTpro reagent is an improvement over the TMTproZero reagent for the accurate quantification of high-charge-state peptides for trigger-based multiplexed assays.