Abstract
Stable isotope labeling of peptides is the basis for numerous mass-spectrometry-based quantification strategies. Isobaric tagging and metabolic labeling, namely, tandem mass tagging (TMT) and SILAC, are among the most widely used techniques for relative protein quantification. Here we report an alternative, precursor-based quantification method using nonisobaric TMT variants: TMTzero (TMT0) and superheavy TMT (shTMT). We term this strategy mass difference tandem mass tagging (mTMT). These TMT variants differ by 11 mass units; however, peptides labeled with these reagents coelute, analogous to SILAC-labeled peptide pairs. As a proof-of-concept, we profiled the proteomes of two cell lines that are frequently used in neuroscience studies, SH-SY5Y and SVGp12, using mTMT and standard SILAC-labeling approaches. We show similar quantified proteins and peptides for each method, with highly correlated fold-changes between workflows. We conclude that mTMT is a suitable alternative for precursor-based protein quantification.