Preparation Of Functionalized Magnetic Nanoparticles And Application In Phosphopeptides

Protein phosphorylation,one of the most important and universal post-translation modifications,plays a vital role in regulating various biological processes,such as cell proliferation,metabolism,differentiation,transcription,and signal transduction.Currently,mass spectrometry?MS?based techniques have been the premier technology for the analysis of protein phosphorylation.Unfortunately,inherent low abundance and ionization efficiency of phosphopeptides make the direct MS analysis still a challenge.Hence,an efficient enrichment process prior to MS analysis is required.In recent years,the development of efficient affinity materials for selective capture of phosphopeptides has attracted wide attention.The selectivity and efficiency for phosphopeptides depend on the enrichment mechanism and the surface structure characteristics of affinity materials.At present,many mesoporous,hybrid or chemically modified affinity materials have been prepared and used for the separation and enrichment of phosphopeptides,including polymer materials,mesoporous materials,monolithic materials,and magnetic nanomaterials.Magnetic nanomaterials have been widely used in many fields,such as medical biology,sensors and catalysis,and sample preconcentration.Compared with traditional adsorbents,magnetic nanomaterials have good magnetic responsiveness,which can be rapidly separated from solution by using an external magnetic field.However,magnetic nanoparticles suffer from several inherent limitations such as aggregation and lack of specific adsorption capacity during the separation process.Therefore,the surface functionalization of magnetic nanoparticles is necessary in practical experiments.In this paper,in order to improve the specific adsorption capacity and stability,magnetic nanoparticles were functionalized to obtain magnetic adsorbents with fast magnetic responsiveness,good biocompatibility,and high selectivity.By combining with MALDI-TOF MS,the prepared materials were used for the separation and detection of phosphopeptides in complex biological samples.The detailed contents were divided into the following five parts:1.Herein,we built an on-chip magnetic system with In₂O₃-coated Fe₃O₄magnetic nanoparticles?MNPs?as solid phase extraction?SPE?adsorbent for the selective enrichment of phosphopeptides by coupling with MALDI-TOF MS.In₂O₃-coated Fe₃O₄ MNPs not only had a shell of indium oxide,giving them a trapping capacity for phosphopeptides,but also enabled easy isolation by positioning an external magnetic field due to their magnetic properties.The on-chip magnetic SPE-MALDI-TOF MS system offered high selectivity for phosphopeptides from standard protein digests,non-fat milk digests,and human serum.Moreover,the designed approach was featured with low detection limit,good reusability,and had great potential for phosphoproteins-related sample analysis.2.We prepared magnetic hydrazide-functionalized poly-amidoamine?PAMAM?dendrimer embedded with TiO₂ for specific enrichment of low-abundance phosphopeptides in complex samples.MNPs were coated with PAMAM dendrimers,after which the terminal groups of PAMAM were functionalized with hydrazide groups.Afterwards,TiO₂ was embedded into the polymer network of hydrazide-functionalized PAMAM MNPs to form the final adsorbent.It was employed to enrich phosphopeptides coupled with MALDI-TOF MS.Electrostatic attraction and hydrogen bonding existed between PAMAM and phosphopeptides while Lewis acid-base interaction was originated between TiO₂ and the targets.The detection limit of?-casein digests was as low as 0.4 fmol.The rapid,efficient,and sensitive method was employed to detect endogenous phosphopeptides in serum samples with satisfactory results.3.In this study,cysteamine hydrochloride-modified chitosan?CYECS?was firstly prepared,after which polyoxometalates?POMs?and CYECS were assembled onto magnetic nanoparticles?MNPs?via a layer-by-layer approach.By combining with MALDI-TOF MS,the material was utilized for effective enrichment of phosphopeptides.The morphologies,zeta potentials,maximum saturation magnetizations,and adsorption capacities for small phosphoric acid molecules of the prepared materials with different layer numbers were investigated.High sensitivity?detection limit of 0.02 fmol?and selectivity toward phosphopeptides were achieved.The recovery of phosphopeptides was determined to be as high as 92.6%.4.In this study,we designed a gadolinium-based immobilized metal ion affinity chromatography material for selective enrichment of phosphopeptides.Gadolinium ions were immobilized onto the surface of glutathione-coated magnetic nanoparticles through a facile and effective synthetic route.The adsorbent integrated the advantages of superparamagnetism of Fe₃O₄ core,good biological compatibility of glutathione,and strong interaction between gadolinium ion and phosphopeptides.A series of experimental parameters of adsorption and desorption steps were investigated.Under the optimum experimental conditions,the selectivity for phosphopeptidesofthepreparedmaterialwasstudiedfromstandard phosphoproteins,the mixture of phosphoproteins and non-phosphoproteins,and real sample.The developed method had high sensitivity,good reusability,and reproducibility.5.Exfoliated Cu/Ga layered double hydroxide?LDH?nanosheets were constructed,over which Fe₃O₄ nanoparticles were uniformly spread to maintain the two-dimensional?2D?morphology.The prepared material was used for the enrichment of phosphopeptides combined with MALDI-TOF MS determinations.The adsorbent has the advantages of high content of metal ion,abundant positive charge,superparamagnetism,large surface area,and good biocompatibility.The composite demonstrated specific capture of phosphopeptides from practical samples,nonfat milk,human saliva,and serum.Fe₃O₄-LDH also had good performance for quantifying abnormally regulated phosphopeptides in leukemia patients sera by using an isotope labeling method.The method gave inspirations of using 2D materials in the field of biomolecules separation.