Research For Enrichment Ability Of Phosphopeptide Using 800TiO₂/Bi/Fe/Zr Composite

Protein phosphorylation,as one of the post-translation modifications,regulates a variety of biological processes,including cell cycle,cell proliferation,apoptosis,DNA damage response and so on.These basic functions are integrated into a single cell to form a complete network.So any minute changes can trigger a series of reactions.such as protein phosphorylation.These cellular signaling networks are more complex in many diseases such as cancer.Therefore,it is helpful to understand the mechanism of disease development and basic cellular function at the system level by studying the whole signal network composed of many phosphoproteinsAlthough protein phosphorylation is universal in cells,its stoichiometry is low and dynamic and it is still major challenges for research on phosphorylation.Presently,it is very common to identify and quantify phosphopeptides with mass spectrometry(MS)-based methods.Given difficult to ionize of phosphopeptide,the application of MS in phosphoproteome is hindered.Therefore,pre-separation and enrichment of phosphopeptides before MS analysis is a critical step.Currently,various methods have been developed to enrich phosphopeptides/protein,including immobilized metal ion affinity chromatography(IMAC),metal oxide affinity chromatography(MOAC),strong anion/cation-exchange chromatography,immunoprecipitation,etc.Among them,the most widely methods are IMAC and MOAC.Taking advantages of the above two methods,the Bi3+;Fe3+ and Zr4+ ions were firstly embedded in TiO2,and a novel nano-materials of 800TiO2/Bi/Fe/Zr were formed by calcining at 800 0 C,then the material is used for phosphopeptides enrichment.The phosphopeptides enrichment ability of 800TiO2/Bi/Fe/Zr composites were firstly estimated using a tryptic peptide mixture of ?-casein(this standard phosphorylated protein containing trace amount of ?-casein).The enrichment specificity of the novel nanocomposite was further evaluated by enrichment of the mixture of tryptic digestion of ?-casein phosphopeptides and BSA in different proportions.Furthermore,HeLa cell lysate have been used to test the ability of the 800TiO2/Bi/Fe/Zr nanocomposite to evaluate its application in complex biological samples.The results show six phosphopeptides were detected with 800TiO2/Bi/Fe/Zr extraction,and the detection limit was 2 × 10-10 M.For semi-complex samples and biological samples,the composite has a good enrichment ability for phosphopeptides.Next,the enrichment capacity was compared between novel nano-composite and TiO2 at three levels:standard phosphopeptides,semi-complex sample and complex biological sample.The results show that not only more abundant phosphopeptides were enriched by novel nanomatrials,but also MS peaks intensity of these phosphopeptides were 7-8 times higher than TiO2 enrichment using simple and semi-complex samples.Inaddition,totally 564 phosphopeptides were identified after enrichment 800TiO2/Bi/Fe/Zr complexes,while totally 345 phosphopeptides were enriched by TiO2.The results show the novel nano-materials had better selective enrichment ability for phosphopeptides than TiO2,indicating that 800TiO2/Bi/Fe/Zr complexes have broad application prospects in phosphorylation related proteomics.