Effective Enrichment Of Phosphopeptides Based On SiO₂ Chromatographic Column Modified By Chiral Asp-based Molecules

Protein phosphorylation is a very common and significant post-translational modification that influences many important biological processes.Conventional mass spectrometry can't detect phosphopeptides directly because of low abundance of organism protein phosphorylation,diversity of phosphorylation and the limitations of analysis technology.Hence,the separation and enrichment of the target proteins/peptides have been attracted much attention in the field of phosphoproteomics.This study concentrated on optimizing IMAC materials and enriching conditions,and respectively researched selective enrichment of phosphopeptides of two enantiomorphism isomers of chiral aspartic acid-based molecules.Two kinds of Asp-based molecules were separately modified on the surfaces of Au sensors on the strength of Au-S.These modified Au sensors were characterized by CA.The interactions between Asp-based molecules and phosphopeptides were investigated by using quartz crystal microbalance?QCM?.The results demonstrated that these Asp-based molecules modified Au sensors could efficiently absorb Serine phosphorylation peptide,especially 2p-Ser and 3p-Ser.Using circular dichroism?CD?,we studied amount of variation of CD signal of acetyl-L?D?-aspartic acid-Fe³⁺and acetyl-L-aspartic acid-Fe³⁺-2p-Ser/3p-Ser when Fe³⁺and 2p-Ser/3p-Ser were added.Acetyl-L?D?-aspartic acid were synthesized and characterized by MS,FT-IR and ¹H-NMR prior to CD measurement.As a result,we suspect that there is a special relationship between CD signal variation and adsorption.The above researches had paved a way for developing suitable materials for the enrichment of phosphopeptides.Therefore,a series of materials were synthesized and characterized by a variety of characterization techniques such as SEM,TG,XPS and BET to confirm that the L?D?-Asp-based molecules had been successfully grafted on to the surface of SiO₂,including SiO₂-L?D?-Asp,SiO₂-L?D?-Asp-OCH₃ and SiO₂-L?D?-Glu-OCH₃.Then,these SiO₂-based m;aterials were applied to enrich mono-and multi-phosphopeptides and the enriching efficiency and selectivity were analyzed by a mass-spectrometric technique.The results obtained from mass spectra indicated that SiO₂-L?D?-Asp materials could well enrich mono-and multi-phosphopeptides under appropriate conditions;SiO₂-L?D?-Asp-OCH₃ and SiO₂-L?D?-Glu-OCH₃ materials could selectively enrich mono-phosphopeptides under identical condition.Furthermore,the enriching efficiency of learus materials was obviously better than dexter-materials.According to the above analysis,those innovative materials will bring a new research opportunity to phosphoproteomics provided that enriching efficiency and selectivity are totally different between learus materials and dexter-materials via adjusting material structures and enriching conditions.