biostudies-literatureGuo YNIGMS NIH HHS101873https://www.ebi.ac.uk/biostudies/studies/S-EPMC9587358biostudies-literatureUnknown9Isobaric 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>i.e.</i>, 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 intMethodsXA benchmarking protocol for intact protein-level Tandem Mass Tag (TMT) labeling for quantitative top-down proteomics.PMC9587358R01 GM118470Cupp-Sutton KALiu XGuo YWu SYu DfalseA benchmarking protocol for intact protein-level Tandem Mass Tag (TMT) labeling for quantitative top-down proteomics.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>i.e.</i>, 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 int2022-01-01T00:00:00Z20222026-05-28T01:20:16.346Z2025-02-18T23:29:48.653ZS-EPMC95873583628127810.1016/j.mex.2022.101873