Dataset Information

Nano-scale solid-phase isobaric labeling for multiplexed quantitative phosphoproteomics

ABSTRACT: We established a workflow for highly sensitive multiplexed quantitative phosphoproteomics using a nano-scale solid-phase tandem mass tag (TMT) labeling reactor. Phosphopeptides were firstly 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 micro-scale reaction 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 nano-scale 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 (http:// jpostdb.org) with the data set identifier PXD025536.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Yasushi Ishihama

PROVIDER: PXD025536 | JPOST Repository | Fri Jul 23 00:00:00 BST 2021

REPOSITORIES: jPOST

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Nanoscale Solid-Phase Isobaric Labeling for Multiplexed Quantitative Phosphoproteomics.

Ogata Kosuke K Tsai Chia-Feng CF Ishihama Yasushi Y

Journal of proteome research 20210722 8

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 enrich ...[more]

PMID: 34292731

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	151218ko04-SepPak-tC18.raw	Raw
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	151218ko07-PLS-2.raw	Raw
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