SMOAC (Sequential Enrichment from MOAC, “Smoke”), a phosphoproteomics strategy for the separation of multiply phosphorylated from monophosphorylated peptides

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Jae Choi, Sergei I. Snovida, Ryan Bomgarden, John C. Rogers
ASMS 2017 Poster
Purpose: We explored sequential methods for complementary phosphopeptide enrichment to find an effective strategy for enrichment of multiply phosphorylated peptides. We also evaluated fractionation strategy with the phosphopeptide enrichment method to show the comprehensive phosphoproteomes. Methods: We implemented a SMOAC strategy. One milligram of Nocodazole-arrested HeLa cell digest sample is our starting sample. We use a titanium dioxide (TiO2) chromatography first for phosphopeptide enrichment. Flow-through and wash fractions from TiO2 were pooled and used for Fe-NTA chromatography for the second enrichment. We subsequently used high pH RP fractionation. We expanded our analytical depth by performing fractionation with pooled phosphopeptide eluates from both TiO2 and Fe-NTA performed in series. Results: The SMOAC strategy enabled effective enrichment of multiply phosphorylated peptides and emphasizes the utility of SMOAC with fractionation to elucidate the comprehensive phosphoproteome.


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