The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry

The success in profiling the phosphoproteome by mass spectrometry-based proteomics has been intimately related to the availability of methods that selectively enrich for phosphopeptides. To this end, we describe a protocol that combines two sequential enrichment steps. First, strong cation exchange (SCX) chromatography separates peptides by solution charge. Phosphate groups contribute to solution charge by adding a negative charge at pH 2.7. Therefore, at that pH, phosphopeptides are expected to elute earlier than their nonphosphorylated homologs. Second, immobilized metal affinity chromatography (IMAC) takes advantage of phosphate's affinity for metal ions such as Fe(3+) to uniformly enrich for phosphopeptides from the previously collected SCX fractions. We have successfully employed the SCX/IMAC enrichment strategy in the exploration of phosphoproteomes from several systems including mouse liver and Drosophila embryos characterizing over 5,500 and 13,000 phosphorylation events, respectively. The SCX/IMAC enrichment protocol requires 2 days, and the entire procedure from cells to a phosphorylation data set can be completed in less than 10 days.