TMTpro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level.

TitleTMTpro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level.
Publication TypeJournal Article
Year of Publication2021
AuthorsJohnson, A, Stadlmeier, M, Wühr, M
JournalJ Proteome Res
Volume20
Issue6
Pagination3043-3052
Date Published2021 06 04
ISSN1535-3907
KeywordsIons, Peptides, Proteomics, Software
Abstract

<p>Multiplexed proteomics is a powerful tool to assay cell states in health and disease, but accurate quantification of relative protein changes is impaired by interference from co-isolated peptides. Interference can be reduced by using MS3-based quantification, but this reduces sensitivity and requires specialized instrumentation. An alternative approach is quantification by complementary ions, the balancer group-peptide conjugates, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with most proteomics instruments. However, complementary ions of the popular TMT-tag form inefficiently and multiplexing is limited to five channels. Here, we evaluate and optimize complementary ion quantification for the recently released TMTpro-tag, which increases complementary ion plexing capacity to eight channels (TMTproC). Furthermore, the beneficial fragmentation properties of TMTpro increase sensitivity for TMTproC, resulting in ∼65% more proteins quantified compared to TMTpro-MS3 and ∼18% more when compared to real-time-search TMTpro-MS3 (RTS-SPS-MS3). TMTproC quantification is more accurate than TMTpro-MS2 and even superior to RTS-SPS-MS3. We provide the software for quantifying TMTproC data as an executable that is compatible with the MaxQuant analysis pipeline. Thus, TMTproC advances multiplexed proteomics data quality and widens access to accurate multiplexed proteomics beyond laboratories with MS3-capable instrumentation.</p>

DOI10.1021/acs.jproteome.0c00813
Alternate JournalJ Proteome Res
PubMed ID33929851
PubMed Central IDPMC8330405
Grant ListR35 GM128813 / GM / NIGMS NIH HHS / United States