Optimization of protein-level tandem mass tag (TMT) labeling conditions in complex samples with top-down proteomics

• Published in: Analytica chimica acta 2022-08, Vol.1221, p.340037-340037, Article 340037
• Main Authors: Guo, Yanting, Yu, Dahang, Cupp-Sutton, Kellye A., Liu, Xiaowen, Wu, Si
• Format: Article
• Language: English
• Subjects: Isobaric labeling; Quantitative proteomics; Tandem mass tags (TMT); Top-down proteomics
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2022.340037

Isobaric chemical tag labels (e.g., iTRAQ and TMT) have been extensively utilized as a standard quantification approach in bottom-up proteomics, which provides high multiplexing capacity and enables MS2-level quantification while not complicating the MS1 scans. We recently demonstrated the feasibility of intact protein TMT labeling for the identification and quantification with top-down proteomics of smaller intact proteoforms ( 9.1, final hydroxylamine concentration >0.3%), and (3) high protein concentration (e.g., > 1.0 μg/μL) resulted in optimal labeling efficiency and minimized production of over/underlabeled side products. >90% labeling efficiency was achieved for E. coli cell lysate after optimization of protein-level TMT labeling conditions. In addition, a double labeling approach was developed for efficiently labeling limited biological samples with low concentrations. This research provides practical guidance for efficient TMT labeling of complex intact protein samples, which can be readily adopted in the high-throughput quantification top-down proteomics. [Display omitted] •Systematic optimization of protein-level TMT labeling in complex sample.•> 90% labeling efficiency with optimal conditions.•Double labeling technique for low concentration samples labeling.