A Multidimentional Phosphopeptide Enrichment Strategy Employing Titanium Dioxide Combined with SIMAC and HILIC for Highly Sensitive Phosphoproteomics

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January 27, 2012

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  • Previously, we optimized Titanium dioxide (TiO2) chromatography as an efficient enrichment method for phosphopeptides only if a “non-phosphopeptide excluder” was introduced in the loading buffer resulting in efficient removal of non-phosphorylated peptides (Larsen MR MCP 2005, Jensen and Larsen RCMS 2007). Later we introduced Sequential elution from IMAC (SIMAC) for the selective separation of mono-phosphorylated peptides from multi-phosphorylated peptides (Thingholm MCP 2008). Here we present a multi-dimensional strategy combining the TiO2 strategy with SIMAC and HILIC separation of the mono-phosphorylated peptides. The strategy is here illustrated for the early interferon gamma (INFg) signaling in rat insulinoma cells and calcium dependent signalling in isolated nerve-terminals. The present strategy provides the highest number of phosphopeptides from low amount of biological material and the cleanest phosphopeptide enrichment seen today, with a routinely yield in separation efficiency of more than 90-95%. It has been applied in our group to a large number of experiments using primary cell material in which limited amount of protein are available including isolated nerve terminals, handpicked Islet of Langerhans and diseased tissue.

    Flow Chemistry

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