A New Method For Selective Enrichment And Mass Spectrometry Identification Of Phosphopeptides Based On Nanomaterials

As an essential post-translational modification,protein phosphorylation can regulate many essential life processes,including cell metabolism,growth,differentiation,and tumor occurrence.Mass spectrometry has been the foremost choice for phosphoproteomics analysis owing to its high sensitivity and accuracy.However,the detection of phosphopeptides using MS directly is still a significant challenge due to the potent inhibitory effects of nonphosphorylated peptides and inorganic salts.Therefore,the isolation and selective enrichment of phosphopeptides from complex biological samples before MS analysis is crucial to phosphoproteome research.To solve the above problem,the following study was conducted in this paper:1.Ti-based IMAC(Immobilized Metal Affinity Chromatography)has shown high potential in phosphoproteome mass-spectrometric analysis.Although the types and structure of Ti-IMAC materials have significantly been developed,most IMAC materials have poor water solubility and limited surface area,which substantially reduce their ability to enrich hydrophilic phosphopeptides selectively.To overcome these two key drawbacks,a novel Ti-based IMAC nanomaterial was prepared by Ti-bonded?-CD anchored on the surface of CNTs(denoted as COOH-CNTs-CD-Ti)is successfully synthesized and applied as a biofunctional adsorbent for selectively enriching trace phosphopeptides.In the selective enrichment process,CNTs provided greater surface area for the absorption of phosphopeptides,while?-CD also offered a more significant opportunity for interaction among phosphopeptides and Ti⁴⁺.COOH-CNTs-CD-Ti with the two properties exhibited higher sensitivity to phosphopeptides even when the concentration of?-casein digests was decreased to 1×10^-11 M,showing a more excellent selective enrichment capability towards complex biological samples.The COOH-CNTs-CD-Ti nanohybrid overcomes two common defects of traditional Ti-IMAC simultaneously based on a material in the process of phosphopeptide enrichment and provides a powerful tool for the pretreatment of phosphoproteome research.2.Specific enrichment of multiphosphopeptides in the presence of nonphosphorylated peptides and monophosphopeptides has been a challenge for in-depth phosphoproteomics.Despite several affinity materials for the enrichment of phosphopeptides before mass spectrometry analysis,the enrichment materials for the multiphosphopeptides are still limited.Meanwhile,most of the reported materials for the multiphosphopeptides enrichment are usually made by multistep strategies,which suffer from the tedious synthetic process and costly raw materials used.To solve the above problems,in this paper,based on the unique properties of multiphosphopeptides,the selective enrichment of multiphosphopeptides was performed by calcium phosphate nanoparticles.Calcium phosphate nanoparticles are synthesized in 10 minutes by two kinds of cheap raw materials at room temperature for specific enrichment of multiphosphopeptides.The results indicated that the nano calcium phosphate exhibited high sensitivity and specificity to multiphosphopeptides.Moreover,the selectivity and limit of the capture method's detection were tested by the tryptic digest mixture of?-casein and bovine serum albumin with the molar ratio of 1:1,1:10,and 1:100.The easy and straightforward synthesis method will conduct to the use of phosphoproteomics research and the validations of the reasons for the specific enrichment of multiphosphopeptides.According to the practical application of phosphoproteome,the novel Ti-IMAC material was first synthesized and used to enrich phosphopeptides.Then,specific enrichment of multiphosphopeptides was performed by calcium phosphate nanoparticles.The paper screened a series of promising enrichment materials to promote the phosphoproteomics research and clarify the influence law of phosphopeptides enrichment efficiency.This work was expected to provide more approaches to prepare more efficient enrichment materials for phosphoproteome research.