Isobaric chemical tag labeling for quantification of intact proteins in complex samples is limited due to the tendency of intact proteins precipitate under labeling conditions and increased sample complexity as a result of side products (i.e., incomplete labeling or labeling of unintended residues). To reduce precipitation under labeling conditions, we developed a technique to remove large proteoforms that allowed for the labeling and characterization of small proteoforms (<35 kDa) using top-down proteomics. We also systematically optimized protein-level Tandem Mass Tag (TMT) labeling conditions to obtain optimal labeling parameters for complex samples. Here, we present a benchmarking protocol for protein-level TMT labeling for quantitative top-down proteomics, including complex intact protein sample preparation, protein-level TMT labeling, top-down LC/MS analysis, and TMT reporter ion quantification.•An optimized protocol for protein-level TMT labeling in complex sample.•Limits production of incorrectly labeled side products for minimization of spectral complexity.•A guideline for isobaric chemical tag quantification in top-down proteomics.
Keywords: Isobaric chemical tag; Protein-level TMT labeling; Quantification; TMT; Top-down proteomics.
© 2022 The Authors. Published by Elsevier B.V.