Novel Reversible Biotinylated Probe for the Selective Enrichment of Phosphorylated Peptides from Complex Mixtures

To improve the detection of phosphorylated peptides/proteins, we developed a novel protocol that involves the chemical derivatization of phosphate groups with a chemically engineered biotinylated-tag (biotin-tag), possessing three functional domains; a biotin group for binding to avidin, a base-labile 4-carboxy fluorenyl methoxycarbonyl (4-carboxy Fmoc) group, and a nucleophilic sulfhydryl moiety on the side-chain of cysteine. Using this approach, the derivatized, enzymatically digested peptides were selectively separated from unrelated sequences and impurities on immobilized avidin. Unlike previously published phosphopeptide enrichment procedures, this approach upon treatment with mild base liberates a covalently bound Gly-Cys analog of the peptide(s) of interest, exhibiting improved RP-HPLC retention and MS ionization properties compared with the precursor phosphopeptide sequence. The results obtained for a model peptide Akt-1 and two protein digests, demonstrated that the method is highly specific and allows selective enrichment of phosphorylated peptides at low concentrations of fmol/μL.