Preparation Of Magnetic Metal Organic Framework Materials And Application In Phosphopeptides Analysis

Protein phosphorylation is a reversible and rapid protein modulation mechanism controlled by kinases,which can directly regulate protein activity.Deviation in the degree and site of phosphorylation can lead to fundamental changes in biological regulation,leading to various diseases.Mass spectrometry(MS)provide a preferred technology for phosphoproteomics analysis.Bottom-up phosphoproteomics analysis typically conducted by ultilizing trypsin to hydrolyze protein into small peptides.Sequences and sites of phosphopeptides are subsequently identified by means of MS.However,dynamic concentration range,complexity of real samples,and low ionization efficiency of phosphopeptides complicate comprehensive analysis of protein phosphorylation,posing significant challenges for direct MS analysis.Therefore,efficient pre-enrichment of low-abundance phosphopeptides in complex systems before MS is the key to achieve large-scale phosphoproteomic analysis.Magnetic solid phase extraction(MSPE)is a novel sample pretreatment technology and has attracted much attention because of its advantages of high adsorption efficiency,simple operation,fast separation speed and low cost.In MSPE,magnetic adsorbents are the key to realize rapid separation and high-efficiency enrichment.Usually,magnetic cores are modified with functionalized molecules to obtain magnetic adsorbents.The presence of magnetic core allows magnetic adsorbents to be rapidly separated with the help of external magnetic field.And functional molecules improve the stability and selectivity of magnetic adsorbents,further enhancing adsorption efficiency.Considering the importance of phosphopeptide enrichment,selecting functional molecules that can specifically recognize phosphate groups for the preparation of magnetic adsorbents greatly enriches the application of MSPE in the separation and enrichment of phosphopeptides.Metal organic frameworks(MOFs)are a type of hybrid materials formed by the connection of metal inorganic subunits and organic ligands containing oxygen or nitrogen donors.MOFs possess highly ordered permanent pore structures,tunable structures,and large specific surface areas,the synergy between metal centers and organic ligands can yield many other properties.Besides,abundant active sites on the surface of MOFs are also beneficial for their post-modification for enhanced performance.Based on these advantages mentioned above,MOFs have been widely used in the field of sample pretreatment.In presenr study,MOFs with unique advantages were served as functional material to modify magnetic core for improved enrichment efficiency.A series of novel magnetic adsorbents integrating the advantages of MOFs and unique magnetic responsiveness from magnetic core were synthesized.The MSPE platform constructed by MOFs-based magnetic adsorbents is combined with MS technology to achieve the enrichment and analysis of phosphopeptide in complex systems.The research works are as follows:1.ZIF-90 functionalized magnetic nanoparticles were prepared,followed by the graft of carnosine via Schiff-base reaction.The synthesized multifunctional material(Fe₃O₄@NH₂@ZIF-90@Car)has the sensitivity of 0.1 fmol and selectivity of 1:500for phosphopeptides enrichment.The feasibility and practicality of as-prepared material were verified by enriching phosphopeptides from skimmed milk,human serum and saliva.2.Bipyridine derivatives were used as organic ligands for self-assembly with metal ions(Zn²⁺)and a visible light-responsive magnetic MOF composite was synthesized.Based on the unique photoresponsive and zwitterionic characteristics of bipyridine derivatives,the synthesized magnetic adsorbent can spectifically capture phosphopeptides by regulating light and p H.The material exhibits good selectivity,repeatability and stability for phosphopeptide enrichment.Finally,it was applied to capture endogenous phosphopeptide from human serum.3.A novel Fe₃O₄@ZIF-8@Zn-Ga LDH enrichment material was prepared by a MOFs-mediated synthesis strategy.Magnetic MOF was used as self-sacrificing template and metal source for LDH generation under the condition of introducing Ga³⁺.The combination of LDH and MOF endows the heterogeneous LDHs with high specific surface area and a large number of exposed affinity sites.The MSPE platform was established by using Fe₃O₄@ZIF-8@Zn-Ga LDH as magnetic adsorbent,and it exhibited good enrichment capacity in phosphopeptide sample.Finally,324 and 122phosphopeptides were identified from human saliva and serum samples,respectively.4.A novel magnetic cationic MOF(Fe₃O₄@ILI-01@Ti⁴⁺)was prepared for phosphopeptide enrichment.Through synergistic electrostatic and hydrophilic interaction,Fe₃O₄@ILI-01@Ti⁴⁺can capture low-abundance phosphopeptides easily.Meanwhile,based on the hydrophilic interaction,glycopeptides can also be selectively enriched.Finally,the material was applied to actual samples to verify its enrichment performance toward phosphopeptides and glycopeptides via LC-MS/MS.The results showed that Fe₃O₄@ILI-01@Ti⁴⁺could effectively enrich the low abundance phosphopeptides/glycopeptides in the complex system.