Figure 10.25.2.

Strategies for the enrichment of phosphopeptides prior to MS analysis. It is possible to perform either IMAC enrichment or TiO₂ enrichment alone, or in combination (SIMAC strategy). Following digestion of proteins, enrichment of phosphopeptides using the SIMAC strategy is achieved first by IMAC enrichment. Some phosphorylated peptides may not be captured by IMAC (IMAC unbound) and should therefore be enriched further by TiO₂. This sample can be analyzed directly by MS/MS. Singly phosphorylated (singly PO₄) peptides are eluted from IMAC (IMAC elution 1), then further enriched by TiO₂ and analyzed by MS/MS. Finally, multiply phosphorylated peptides are eluted from IMAC and analyzed directly by MS/MS.