Development And Application Of Novel Material And Approach For Enrichment Of Phosphopeptides

Phosphorylation,as one of post-translational modifications,play important role in the life process,including proliferation,development and differentiation and so on.With view to the importance of protein phosphorylation,it is necessary to identify and analyze the phosphorylation in detail.However,low abudance of phosphorylated protein and its versatility in vivo challenge the identification of phosphorylation based on mass spectrometry?MS?.In addition,phosphate group loss may occurs during the sample treatment.Therefore,enrichment and appropriate treatment of phosphopeptides/proteins before mass spectrometric analysis are crucial for the phosphorylation identification.In other words,efficient enrichment of phosphopeptides would improve their identification efficiency significantly.Presently,immunoaffinity purification,metal cation adsorption,and ion change chromatography have been developed for this purpose.It is still need to develop new strategy for the enrichment of phosphopeptides due to the complexity of proteomes in biological samples.In this study,a TiO₂/Bi/Fe/Zr nanocomposite for phosphopeptides enrichment was synthesized by sol-gel method and characterized.Asthe result,transmission electron microscope?TEM?and scanning electron microscope?SEM?images showed that this material is near spheric,shape,with a particle size around 50 nm.Further X-ray photoelectron spectroscopy?XPS?,TEM mapping and energy dispersive X-ray spectroscopy?EDX?spectra showed the existence of Ti,Zr,Bi and Fe.The obvious peaks of Bi in XPS image suggested that Bi is presented as a separate phase on the surface of TiO₂ whereas the peaks of Fe and Zr are rarely observed,implying that Fe and Zr mainly exist inside the TiO₂ particles since the similar radius of Fe and Zr with Ti.The N₂ adsorption–desorption isotherm test indicated that,due to the doping of Bi³⁺,Fe³⁺and Zr⁴⁺,the novel nanocomposite?13.54 cm²/g?has a higher specific surface area than that of the unmodified TiO₂?5.07 cm²/g?,indicating that the novel material could provide more adsorption sites for phosphopeptides.After optimization,the experimental conditions for the enrichment of phosphopeptides was as follows:loading buffer,1 M glycolic acid,5%trifluoroacetic acid?TFA?and 80%acetonitrile?ACN?;washing buffer,30%ACN,1%TFA.Then,the adsorption capacity of TiO₂/Bi/Fe/Zr nanocomposite for phosphopeptides was evaluated by using the tryptic peptides from?-casein,and the mixture of?-casein with BSA at different ratio.As the result,when the ratio of tryptic peptides from?-casein against BSA was 1:100,TiO₂/Bi/Fe/Zr nanocomposite could enrich six phosphopeptides of?-casein whereas commercial TiO₂ could enrich only three phosphopeptides.When the ratio was 1:1000,the number of phosphopeptides enriched by TiO₂ was zero but there was two for TiO₂/Bi/Fe/Zr nanocomposite.Finally,HeLa cell lysate was used to evaluate the application of this nanocomposite in biological samples.From HeLa cell lysate,totally 1276 phosphopeptides containing 1887 phosphorylation sites weresuccessfullyidentifiedafterenrichmentby,TiO₂/Bi/Fe/Zr nanocomposite,compared to 1076 phosphopeptides comprising 1650phosphorylation sites after TiO₂ enrichment.It indicated that the TiO₂/Bi/Fe/Zr nanocomposite have high capacity in the enrichment of phosphopeptides,implying a potential application in phosphoproteomics.Furthermore,TiO₂/Bi/Fe/Zr nanocomposite was used to explore phosphoproteome of rat liver sinusoidal endothelial cell?LSEC?plasma membrane after partial hepatectomy.After enriched by TiO₂/Bi/Fe/Zr nanocomposite,totally 133 phosphopeptides corresponding to 98phosphoproteins were identified in LSECs plasma membrane.Among them,87 and 79 phosphopeptides corresponding to 69 and 68 phosphoproteins were identified from the sham and partial hepatectomy?PHx?72 h groups,respectively.Among the 98 phosphoproteins,39 proteins were identified from both sham and PHx72 groups,and 24 proteins have obviously changed expression levels?at least 1.2-fold change?.According to KEEG analysis,these phosphoproteins are mainly involved in insulin and adenosine5‘-monophosphate?AMP?-activated protein kinase?AMPK?signaling pathways which are closely related to angiogenesis.In brief,this study was not only expands the application of this novel material,but also provide clues for the study of angiogenesis in liver regeneration.To further improve the specificity of TiO₂ for phosphopeptides,TiO₂/Si/Yb nanomaterial was synthesized by sol-gel method and characterized.TEM image and the N₂ adsorption–desorption isotherm test suggested that,similar to TiO₂/Bi/Fe/Zr nanocomposite,TiO₂/Si/Yb nanomaterial is approximately spheric,and the particle size is around 13 nm.Incorporation of silicon in this material could increase the thermal stability of TiO₂,prevent the over-growth of grain,thus the surface-to-volume of the material was raised from 10.8 m²/g to 102.1 m²/g.As the result,the TiO₂/Si/Yb nanomaterial have more adsorption sites for phosphopeptides.Meanwhile,Yb³⁺doping could enhance the electrostatic adsorption capacity for phosphopeptides.The XPS,mapping image and EDS spectrum showed the existence of Ti,Si and Yb elements,indicating that TiO₂/Si/Yb nanocomposite was synthesized successfully.Similarly,the selective enrichment capacity of TiO₂/Si/Yb nanomaterial was firstly tested on the tryptic peptides from?-casein,then the mixture of?-casein and BSA at different proportions.The results revealed that both quantity and background noises of enrichment phosphopeptides,TiO₂/Si/Yb nanomaterial is superior to that of commercial TiO₂.Moreover,TiO₂/Si/Yb has lower detection limit of phosphopeptides?4?10^-1111 M?than that of commercial TiO₂?2?10^-99 M?.Next,the digestions of nonfat milk and human serum were used to test the application of the TiO₂/Si/Yb.In total,ten phosphopeptides from trypsin digested nonfat milk were clearly observed after enrichment by TiO₂/Si/Yb nanomaterial,compared to eight phosphopeptides by that of commercial TiO_2.For trypsin digested human serum,four phosphopeptides were easily detected whereas three phosphopeptides were enriched by TiO₂.Whether for nonfat milk or for human serum sample,phosphopeptides enriched by TiO₂/Si/Yb exhibit clean background in MS spectrum.These results demonstrated that the TiO₂/Si/Yb nanomaterial has higher capacity for the selective enrichment of phosphopepetides as compared with commercial TiO₂.