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Title: Nanoscale Solid-Phase Isobaric Labeling for Multiplexed Quantitative Phosphoproteomics
Authors: Ogata, Kosuke  kyouindb  KAKEN_id  orcid (unconfirmed)
Tsai, Chia-Feng
Ishihama, Yasushi
Author's alias: 小形, 公亮
石濱, 泰
Keywords: TMT labeling
solid-phase reactor
titanium oxide chromatography
multiplexed phosphoproteomics
Issue Date: Aug-2021
Publisher: American Chemical Society (ACS)
Journal title: Journal of Proteome Research
Volume: 20
Issue: 8
Start page: 4193
End page: 4202
Abstract: We established a workflow for highly sensitive multiplexed quantitative phosphoproteomics using a nanoscale solid-phase tandem mass tag (TMT) labeling reactor. Phosphopeptides were first enriched by titanium oxide chromatography and then labeled with isobaric TMT reagents in a StageTip packed with hydrophobic polymer-based sorbents. We found that TMT-labeled singly phosphorylated peptides tend to flow through the titanium oxide column. Therefore, TMT labeling should be performed after the enrichment step from tryptic peptides, resulting in the need for microscale reactions with small amounts of phosphopeptides. Using an optimized protocol for tens to hundreds of nanograms of phosphopeptides, we obtained a nearly 10-fold increase in sensitivity compared to the conventional solution-based TMT protocol. We demonstrate that this nanoscale phosphoproteomics protocol works for 50 μg of HeLa proteins treated with selumetinib, and we successfully quantified the selumetinib-regulated phosphorylated sites on a proteome scale. The MS raw data files have been deposited with the ProteomeXchange Consortium via the jPOST partner repository ( with the data set identifier PXD025536.
Rights: This document is the Accepted Manuscript version of a Published Work that appeared in final form in 'Journal of Proteome Research', copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see 10.1021/acs.jproteome.1c00444.
The full-text file will be made open to the public on 22 July 2022 in accordance with publisher's 'Terms and Conditions for Self-Archiving'
This is not the published version. Please cite only the published version. この論文は出版社版でありません。引用の際には出版社版をご確認ご利用ください。
DOI(Published Version): 10.1021/acs.jproteome.1c00444
PubMed ID: 34292731
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