Enhancement of ionization efficiency and selective enrichment of phosphorylated peptides from complex protein...

To improve the detection of phosphorylated peptides/proteins, a combination of optimized MS-based strategies were used involving chemical derivatization with a polyhistidine-tag (His-tag) and affinity enrichment of the resulting His-tag peptides on a nanoscale Ni(2+)-IMAC column. The phosphoserine and phosphothreonine peptides were derivatized using a one-pot beta-elimination/Michael addition reaction with a reversible His-tag possessing a thiol-containing Cys residue. The His-tag peptides were enriched selectively by Ni(2+)-IMAC and released using either imidazole or cleavage with Factor Xa. This novel capture and enzyme-mediated release provided an additional element of selectivity and yielded phosphopeptide-specific modifications with enhanced MS ionization characteristics. The eluted peptides were mapped using MALDI-TOF MS and QTRAP ESI-MS/MS techniques. The results obtained for a model peptide and two tryptic protein digests show that the method is highly specific and allows selective enrichment of phosphorylated peptides at low concentrations of femtomoles per microliter.