Phosphopeptide Enrichment

An important step to most MS based phosphoprotein analyses is the affinity enrichment of the phosphopeptides. Currently, the MS & Proteomics Resource uses 2 main approaches. The first approach is based on the use of strong cation exchange chromatography (SCX) to enrich and fractionate for early eluting phosphopeptides. This is most applicable to very large sample amounts (i.e. 50µg to 1mg) and is described in Large Scale Phosphoprotein profiling. The second approach has been highly utilized due to its robustness and uses TiO₂ as the affinity support. Both of these approaches can be combined for a more effective phosphopeptide enrichment. The use of anti-phosphotyrosine antibodies is a third choice for enrichment of phosphotyrosine containing proteins and peptides. The specific method of choice will be discussed with the individual investigator for optimum experimental outcome.