Preparation Of Multiple Composition And Dimensional Magnetic Composite Nanomaterials And Their Applications In Phosphoproteomics

Protein is an important component of all cells and tissues of the human body.Many specific functions of the living organisms are realized through protein.Most proteins are modified by the covalent binding of various functional groups（such as methyl,acetyl,glycosyl and phosphoryl groups）during the complicated process from transcription to translation,resulting in more than 300 post-translation modifications（PTMs）.Phosphorylation is one of the most common post-translational modifications of proteins.Protein phosphorylation is the process of modifying phosphate groups on specific amino acids（mainly serine,threonine and tyrosine）catalyzed by protein kinases.It has been estimated that nearly 30%of the proteins in eukaryotic cells are phosphorylated.Reversible protein phosphorylation plays a vital role in regulating multiple biological processes including signal transduction,metabolism,cell growth,adhesion and migration.Abnormal phosphorylation is associated with various diseases,such as cardiovascular disease,cerebrovascular diseases,Parkinson’s disease,Alzheimer’s disease,diabetes and cancer.Therefore,the identification and analysis of phosphoproteins is great significance for researching the phosphoproteomics-related biological and pathological processes,as well as the prevention and early diagnosis of related diseases.Mass spectrometry（MS）with high sensitivity and reliability has become the most essential analysis method for phosphoproteins.The analysis of phosphoproteins usually uses phosphopeptides after digest for MS detection.However,the direct detection of phosphopeptides by MS is difficult due to the low stoichiometry(usually 10^-9 M),poor ionization efficiency and the significant interference derived from abundant non-phosphopeptides（or single phosphopeptides against multiple phosphopeptides）.Therefore,effective separation and enrichment of phosphopeptides prior to MS analysis is the prerequisite to research on phosphoproteomics.Magnetic nanoparticles with excellent magnetic responsiveness,biocompatibility,low toxicity and special targeting properties have been widely applied to magnetic resonance imaging,tumor therapy,targeted drug carriers,gene therapy and biological separation.Developing novel magnetic composite nanomaterials for the simple and efficient enrichment of phosphopeptides has become the phosphoproteomics research hotspot since magnetic composite nanomaterials can greatly simplify the separation process,reduce the loss of materials and samples through magnetic separation,as a result,increasing the enrichment efficiency and reusability.In this paper,a series of new magnetic composite nanomaterials are systematically developed for enrichment of phosphopeptides.The main contents are summarized as follows:1.PAMAM-PMAA brush-functionalized magnetic composite nanospheres for selective enrichment of phosphopeptidesFirstly,the supermagnetic Fe₃O₄ cores were synthesized through a solvothermal method and modified with PDA through dopamine selfpolymerization procedures in order to promote the polymer coating in the next step.Then,PMAA brushes with abundant carboxyl groups were introduced to the surface of the nanospheres by the SI-ATRP technique.Finally,PAMAM dendrimer containing abundant amine groups was grafted to each PMAA chain through amide reaction to obtain the Fe₃O₄@PDA@PMAA@PAMAM nanospheres for enrichment of phosphopeptides based on the interaction between amino group and phosphate group.The prepared Fe₃O₄@PDA@PMAA@PAMAM nanospheres have uniform morphology,good dispersion and remarkable magnetic responsiveness(42.4 emu g^-1,separation time less than 20 s).The well-defined structure and abundant amine groups of PAMAM dendrimer endowed the nanospheres with excellent performance and tunable bonding strength for phosphopeptides.Specific enrichment of phosphopeptides with different phosphorylation sites are achieved by simply modulating the polarity and acidity of the loading and elution conditions.In the enrichment experiment of model protein digestion solution,the magnetic nanospheres showed high detection sensitivity（1fmol/μL）,excellent selectivity（β-casein/BSA=1:500）and good reusability（5 times）.Meanwhile,the enrichment performance of the magnetic nanospheres was also investigated in biological samples（such as skim milk,human saliva）.2.Preparation of different dimension Ti₃C₂T_x@PAMAM@Fe₃O₄ composite materials and their phosphopeptide enrichment performanceThe limited specific surface area of the magnetic nanosphere and tedious preparation process decrease the enrichment efficiency towards phosphopeptides to some extent.Ti₃C₂T_x MXene,a new two-dimension nanomaterials with large specific surface area and abundant functional groups,have been used to load PAMAM dendrimer and magnetic Fe₃O₄ nanoparticles to prepare Ti₃C₂T_x@PAMAM@Fe₃O₄materials through electrostatic interaction.The preparation method of Ti₃C₂T_x@PAMAM@Fe₃O₄ materials is very simple without the use of binding reagents,which greatly decrease the consume of time and cost.Due to the tunable distance between layers,the two-dimensional and three-dimensional Ti₃C₂T_x@PAMAM@Fe₃O₄composites were synthesized simultaneously.In addition,the enrichment experiment results from both model protein（α-andβ-casein）and complex biological samples（nonfat milk,saliva,serum and rat brain lysate）revealed that the enrichment performance is dimension-dependence.The 2-D Ti₃C₂T_x@PAMAM@Fe₃O₄material is applied to selective enrichment of phosphopeptides,which showed high sensitivity(10^-9 M)and excellent selectivity（1:800）.As a result,1705 phosphopeptides were successfully identified from the rat brain lysate.3.In-situ preparation and regulation of two-dimensional magnetic Fe₃O₄/Ti O₂@Ti₃C₂T_x MXene composites nanomaterials for high efficiency enrichment of phosphopeptidesThe above affinity materials enrich phosphopeptides mainly depending on the electrostatic and hydrogen bonding interaction between amine and phosphate groups.The materials with single affinity sites tend to enrich one kind of phosphopeptides,resulting in the lost of some phosphopeptides information.Besides,the combination between functional molecular and substrate is weak based on electrostatic interaction.Hence,we proposed a mild one-pot method to prepare magnetic Fe₃O₄/Ti O₂@Ti₃C₂T_xcomposite nanomaterials by in-situ oxidation of Ti₃C₂T_x MXene and simultaneous growth of Fe₃O₄based on the structure and composition merits of Ti₃C₂T_x MXene.The two-dimensional magnetic Fe₃O₄/Ti O₂@Ti₃C₂T_x composite nanomaterials have the advantages of uniform morphology,stable structure,simple synthesis process and good magnetic response.The multi-affinity sites（Ti-O,Fe-O and amino group）of the composite nanomaterial endowed the materials with excellent enrichment performance with high sensitivity（0.1fmol/μL）,excellent selectivity（β-casein:BSA=1:5000,molar ratio）,good repeatability（5 times）and high enrichment capacity（200 mg/g）.Moreover,the composite nanomaterials also showed excellent enrichment performance towards complex biological samples（such as nonfat milk,human saliva,human serum and rat brain lysates）.1420 phosphopeptides（including 1020monophosphopeptides and 219 multiphosphopeptides）were successfully identified from rat brain lysates after enrichment by the materials,showing attractive application prospect.