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acs.jproteome.1c00444.pdf | 1.95 MB | Adobe PDF | 見る/開く |
タイトル: | Nanoscale Solid-Phase Isobaric Labeling for Multiplexed Quantitative Phosphoproteomics |
著者: | Ogata, Kosuke https://orcid.org/0000-0002-0634-3990 (unconfirmed) Tsai, Chia-Feng Ishihama, Yasushi https://orcid.org/0000-0001-7714-203X (unconfirmed) |
著者名の別形: | 小形, 公亮 石濱, 泰 |
キーワード: | TMT labeling solid-phase reactor phosphopeptides titanium oxide chromatography multiplexed phosphoproteomics |
発行日: | Aug-2021 |
出版者: | American Chemical Society (ACS) |
誌名: | Journal of Proteome Research |
巻: | 20 |
号: | 8 |
開始ページ: | 4193 |
終了ページ: | 4202 |
抄録: | 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 (https://jpostdb.org) with the data set identifier PXD025536. |
著作権等: | 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 https://doi.org/ 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。 |
URI: | http://hdl.handle.net/2433/265265 |
DOI(出版社版): | 10.1021/acs.jproteome.1c00444 |
PubMed ID: | 34292731 |
出現コレクション: | 学術雑誌掲載論文等 |
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