Post-translational modification phosphopeptides characterization via Free Radical–Initiated Peptide Sequencing (FRIPS) Mass Spectrometry

Mass spectrometry-MS based proteomics is a developing field that holds powerful insights into probing accurate structure characterization while allowing for quantiation. Post-translational modifications (PTMs) play significant roles in the regulation of numerous biological processes. Specifically, our group is interested in studying phosphopeptides which is perhaps one of the most frequent PTMs. Our groups aims to achieve a novel, high-throughput analytical method for phosphopeptides identification, structural characterization, and quantitation which has previously been demonstrated to be difficult. Amino acid residues such as serine, threonine, and tyrosine are some of the most common peptides to become phosphorylated. Free Radical Initiated Peptide Sequencing (FRIPS) reagents have been synthesized previously in our group with a components of a peptide coupling site, a pyridine moiety with a high proton affinity, and a TEMPO-precursor site. The coupling site derivatizes at the C-terminus of peptides, the pyridine moiety helps maintain a fixed positive charge in the mass spectrometor, and upon initial collision induced dissociation the TEMPO-precursor site forms the nascent free radical. Fragmentation of the phosphopeptides are observed by MS/MS and MS/MS/MS. Initial studies show full amino acid residue fragmentation as well as the intact phosphate group.