Reproducibility of label-free single-shot phosphoproteomics applied to CRC cell lines

There is a need for robust phosphopeptide enrichment methods to allow signaling network analysis in cancer cell lines and tissues with minimal fractionation. With recent instrument developments thousands of unique phosphopeptides can be detected by single-shot LC-MS/MS. However, successful phosphoproteomics experiments still rely on efficient phosphopeptide enrichment from a tryptic digest prior to LC-MS/MS analysis. Here we describe a performance assessment of HAMMOC (hydroxyl acid modified metal affinity chromatography) (Sugiyama MCP2007, Kyono, JPR 2008) combined with single shot label-free quantitation at 500 µg peptide input level. In a triplicate analysis we observe good phosphopeptide identification reproducibility (75.8%), depth of identification (6014-6150 phosphopeptides) and reproducibility of label-free quantification (CV 17.8%, Pearson r 0.87-0.98) by single-shot LC-MS/MS.