Nanoscale Solid-Phase Isobaric Labeling for Multiplexed Quantitative Phosphoproteomics

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.