Post-translational Modification Of Peptide And Protein Signals In The Mass Spectrometry Sensitizing Study

The post-translational modifications (PTMs) of proteins is a common biological mechanism for regulating protein localization, function, and turnover. A large number of different modifications are reported, and among these modifications, phosphorylation is the most widespread and impotant. It plays critical roles in the regulation of many cellular processes including: cell cycle, growth, apoptosis, and differentiation. Thus, the identification and characterization of phosphorylation sites is crucial for the understanding of various signaling events.Low-stoichiometry of modification events and the low ionization efficiency of phosphopeptides under standard matrix-assisted laser desorption/ionization mass spectrometry conditions compared with their non-phosphorylated analogs may prevent the detection of specific phosphopeptides.Chemical modification strategies based on the alkali-mediatedβ-elimination of phosphoric acid from phosphoserine or phosphothreonine sites followed by Michael addition are increasingly being described for such analyses. We introduced some thiol and amino-group contained affinity labels and in our present work aβ-elimination/Michael addition was used to replace the phosphate groups of pSer or pThr by a group which gives rise to an enhanced ionization efficiency.We synthesized some phosphorylated serine and threonine-containing kemptides as model peptides. In order to find optimum reaction conditions and nucleophile,β-elimination/Michael addition was examined using these phosphorylated model peptides and phosphorylated model proteinα-casein. And this method is also examined using glycosylated model peptides.Derivatization of phosphorylated peptides with S- and N-nucleophiles enhanced the ionization efficiency in MALDI-TOF MS. Michael addition with ethanethiol resulted in products showing higher matrix-assisted laser desorption/ionization signal intensities compared with those of the corresponding phosphorylated precursors. Whereas complete elimination of phosphate could be achieved by treatment with barium hydroxide in water, the yield of the Michael adduct strongly depended on the nucleophile and the peptide sequence.Theβ-elimination/Michael addition reaction used to replace the phosphate moiety of phosphoserine or phosphothreonine peptides by the nucleophiles which should give rise to enhanced ionization efficiency and signal intensities in mass spectrometry, then sequencing of the peptide and assignment of the phosphorylation site can be more simple.