Novel Magnetic Nanocomposites Developed For The Application In Separation And Analysis Of Proteins/Peptides

Many low-abundance endogenous peptides in biological fluids or tissues may provide valuable information for the detection or interpretation of many diseases or pathology,which are potential biomarkers of high specificity and clinical sensitivity.It is still a great challenge to directly detect low-abundance peptides from complex biological matrix by mass spectrometry(MS)due to their extremely low levels.Because of their large specific surface area,abundant active affinity sites and unique magnetic properties,magnetic nanomaterials have attracted extensive attention in the separation and enrichment of low-abundance peptides/phosphopetides,and become a research hotspot in the separation,enrichment and identification of proteomics/peptidomics.Numerous researches have been developed to solve the problems of low concentration,low ionization efficiency and signal suppression of high abundance proteins/peptides in mass spectrometry during the analysis of low-abundance peptides/phosphopeptides.In this work,several novel magnetic affinity nanomaterials were designed and developed for capture of low-abundance peptides and selectively enrichment of phosphopeptides by a simple,fast and efficient magnetic extraction process.Furthermore,the magnetic affinity nano-/micro-probes were employed to separate and enrich of low-abundance peptides and phosphopeptides in real biological samples,and good results were obtained through a bio-mass spectrometry method.1.We gave an introduction of the components of a bio-mass spectrometry instrument and a detailed review on the advances in the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS)technology,containing the history,the working principle and the choice of matrix.In addition,the significance of both proteomics and peptidomics researches and the strategy of separation and enrichment of low-abundance peptides and phosphopeptides were exposited.Then,we summarized the functional magnetic materials for separation and enrichment of low-abundance peptides and phosphopeptides in recent years.Finally,the main study ideas of this thesis were proposed briefly.2.Low-abundance peptides play an important role in indicating the physiological status of the body and can be especially analyzed for clinical diagnosis.Hybrid magnetic core-shell porous carbon nanocomposites(Co/Ni-MCN),which were synthesized in a facile and simple one-step approach using bimetallic MOF(Co/Ni-ZIF)as precursors,were applied in the separation and analysis of low-abundance peptides for the first time.At first,the condition of the reaction was optimized,so that Co/Ni-ZIF crystals,the precursor,which have two kinds of regular morphology,could be prepared at ambient temperature,using Co and Ni as the metal center and 2-MIM as the organic ligand.Subsequently,the magnetic MOF-derived nanoporous carbon materials were acquired through thermal treatment of Co/Ni-ZIF crystals under a nitrogen atmosphere.The physicochemical properties of the prepared Co/Ni-ZIF and Co/Ni-MCN materials were thoroughly characterized by SEM,TEM,XRD,EDS,SQUID,etc.Then,the obtained Co/Ni-MCN materials were applied in the research of the separation and effecient enrichment of low-abundance peptides.3.A novel hydrophilic guanidyl-functionalized magnetic bimetallic metal-organic frameworks composites(denoted as Fe₃O₄@Hf/Ti-MOF-Gua)were designed and sythesized by a facile postsynthetic method.Briefly,Hf/Ti-MOF-NH₂was firstly coated on the surface of Fe₃O₄ core by selecting Hf and Ti as the metal center,and 2-aminoterephthalic acid as the organic ligand.Then,guanidyl group was introduced to the affinity site.Owing to the abundant Hf⁴⁺/Ti⁴⁺affinity sites on the surface,the probe possesses high affinity towards the phosphopeptides.In addition,the polymer shell endows the magnetic composites with large amount of guanidyl groups,which is beneficial to enhance the affinity interaction between phosphopeptides and the material.Thus,Fe₃O₄@Hf/Ti-MOF-Gua affinity probe exhibited high selectivity toward phosphopeptides from human saliva.4.A new promising strategy to isolate and enrich global-/mono-phosphopeptides from biological samples was proposed.A facile and simple solvothermal method was developed for the synthesis of magnetic nickel-based iron oxide nanocomposites(MNFOs)with different ratios of Ni²⁺to Fe³⁺for different reaction time.Two factors including dosage of Ni source and length of reaction time were investigated for the influence on the morphology and composition of MNFOs,as well as their distinct selectivity for different phosphopeptides.After thorough characterization,the possible formation mechanism of MNFOs was proposed.Very interestingly,MNFOs with Ni²⁺/Fe³⁺ratios of 4:5 prepared for 8 h(MNFO-S)and for 24 h(MNFO-L)can selectively capture global-and mono-phosphopeptides,respectively,at fmol level.These two affinity probes have been exploited to isolate and enrich the phosphopeptides from human normal hepatic cells HL 7702 after exposure to atmospheric fine particulates(PM_2.1),which revealed that the protein phosphorylation level was increased significantly in cells after simulation by fine particulate matters.5.The iron oxide nanoparticles(Fe₃O₄)were prepared by organic molecule-assisted method in aqueous solution.The facile synthetic process of Fe₃O₄nanoparticles was conducted only by mixing Fe Cl₂ and 2-methylimidazole(2-MIM)without any additives.A possible growth mechanism of the Fe₃O₄ nanocrystals was proposed for this mild reaction.Then,the Fe₃O₄ nanoparticles were anchored onto graphene oxide(GO)sheets in water by ultrasound-assisted method,and an affinity probe with strong biocompatibility was developed for sequential enrichment of low-abundance peptides and phosphopeptides.Due to the hydroxy and carboxylic groups of GO sheets,Fe₃O₄/GO probe exhibits excellent performance for enriching low-abundance hydrophilic peptides,while the Fe₃O₄ nanoparticles endure the probe with specific affinity to phosphopeptides.Consequently,the prepared Fe₃O₄/GO material possesses excellent feature as a sequent multifunctional affinity probe for low-abundance peptides and phosphopeptides from complex biological matrices,which could be detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.