Mass Spectrometry Analysis And New Methods For Targeted Capture Of Active Biomolecules

Amino acids,peptides,proteins,and glycans,which with biological activity play an important role in regulating various physiological and pathological processes.The accurate identification of their structures and the precise monitoring of their quantity can help clarify their mechanisms of action in biological systems and medical diagnosis,which have a profound influence.With the development of molecular biology,and analytical technology,mass spectrometry has shown obvious advantages in proteomics,medical diagnosis,and other fields due to its advantages of accurate molecular weight determination,high sensitivity and low sample consumption.However,many highly active biomolecules with low natural abundance are not easy to be detected by mass spectrometry.Therefore,it is particularly important to design new efficient and specific functional enrichment materials for targeted enrichment and detection of low-concentration target biomolecules.In view of this,in this paper,the derivatization method was combined with biological mass spectrometry to identify the phosphoryl peptide libraries and eight disaccharide isomers.Three novel IMAC functional materials were designed and synthesized successfully.Furthermore,we accomplished the targeted enrichment of bioactive molecules such as bioactive peptides and metal-binding proteins by combining the qualitative and quantitative properties of the mass spectrum.The urine sample and complex biological samples from patients with the disease were applied to verify the feasibility of these new technologies and methods.As a result,it achieved some innovative research and has impressive application prospects in clinical research.The main research work is as follows:1)A library of phosphoryl oligopeptides was synthesized rapidly by a one-pot method.In this process,unprotected amino acids reacted with phenyl ethyl phosphonate,which played the role of organophosphorus-inducing reagent.The result of UPLC-MS showed that the conversion rate of amino acid was as high as 78%.The MS/MS results of the derivative products for twenty L-type amino acids showed that the length of the phosphoryl peptide chain was greatly affected by the properties of the amino acids.At the same time,it was less affected by the steric hindrance of the side chain.In addition,through the results of multi-level mass spectrometry,it was found that the peptide chain was fragmented from the C-terminus to the N-terminus and then formed A-type and B-type fragment ions in the positive ion mode.And with the increase of the length of the phosphoryl peptide chain,the fragmentation patterns became more obvious.However,the phosphoryl reagent and the amino acid side chain were fragmented easily in the negative ion mode.And the MS/MS spectrum became more complicated,which needed to be further studied.2)Eight disaccharide isomers were derivated with amino acids.The tandem mass spectrometry results of[M+H]⁺ions for the products above showed that the species and intensities of the fragment ions differed with the disaccharide structure.The results displayed that only arginine could trigger the cleavage of the sugar backbone by screening the twenty amino acids.Then eight disaccharide isomers were derivatized using arginine as a derivatizing reagent,and the results showed that there existed significant differences in the MS/MS spectra of the derivatized products for different disaccharides isomers.The strategy can be successfully applied to identify the isomeric structures of eight disaccharide isomers.3)A novel immobilized metal affinity chromatography（IMAC）functional composite,mNi@N-GrT@PDA@Ti⁴⁺,was prepared based on ultrathin magnetic nitrogen-doped graphene tube（mNi@N-GrT）after chelated Ti⁴⁺with polydopamine,following as a magnetic solid-phase extraction sorbent for rapidly selective enrichment and mass spectrometry identification of phosphorylated peptides.After the optimization of the enrichment parameters,the composite exihibited high specificity in the pre-enrichment of phosphopeptides from a digest mixture ofβ-casein and bovine serum albumin（BSA）with a different ratio.The robost method presented the low detection limits(1 fmo L L^-1)and excellent selectivity for phosphopeptides,which can be determined even in the mixture of casein and BSA with a molar ration of even 1:100.Furthermore,the selective enrichment of phosphopeptides in the complex biological samples,such as mouse brain and Hela cells,was successfully carried out.The results showed that 2087 phosphorylated peptides were identified in the complex samples,respectively.The enrichment performance of mNi@N-GrT@PDA@Ti⁴⁺was satisfactory,suggesting that this composite provided a potential application in the phosphoproteomic.4)A novel core-shell MOF composite,SiO₂@50Benz-Cys,was synthesized by a solvent-assisted ligand exchange strategy（SALE）.In light of its excellent affinity with bioethanol small molecules after chelated with mercury ions,the composite can successfully targeted enrich the active amino acid molecules of the total glutathione（GSH）and total homocysteine in urine as an S-Hg-S manner with good biocompatibility.By optimizing the factors on the enrichment efficiency,such as the pH value of the sample,the concentration of DTT in the eluent and the amount of composite,a LC-MS method for the detection of total GSH and total Hcy was established,with the detection limits of 0.5 and 1 nmol L^-1 for GSH and Hcy,respectively.And the recoveries of GSH and Hcy were in the range of 85.3-105%and79.5-103%,which showed good precision and accuracy.The strategy was further applied to the quantitative determination of GSH and Hcy in the urine of healthy group,immunoglobulin A vasculitis（IgAV）and immunoglobulin A vasculitis with nephritis（IgAVN）patients.It was found that the concentrations of GSH and Hcy were significantly different between the healthy group and patient from the analysis of variance（ANOVA）.Benefiting from the matrix interference elimination of the biological samples in the process of extraction,this methodology established did not require special derivatization of GSH and Hcy,together with the advantages of convenience,rapidity,wide linear range,and high sensitivity.It provides a better understanding of the concentration alterations for GSH and Hcy in IgAVN patient body and the evaluation of the oxidative stress state in vivo,besides a further reference for clinical research.5)A 3D ordered micropore and mesoporous composite,HKUST-1@DTMZ with the confinement effect was constructed as an affinity material for mercury metal proteins identification via an IMAC strategy for the specific enrichment of mercury-binding protein in the membranous nephropathy patient urine.The potential of this IMAC material for targeted capture of mercury-binding proteins was evaluated by applying human serum albumin and bovine serum albumin as models.The feasibility of this method for predicting mercury-binding proteins was verified by means of SDS-PAGE and MALDI-TOF-MS techniques.Combined IMAC technology with mass spectrometry,the mercury-binding proteins in the urine of patients with membranous nephropathy（n=15）were selective captured and determined further through the in vivo simulation of SD rats.The experimental results showed that a total of twenty mercury-binding proteins.The enriched metal-binding proteins were mainly involved in some biological processes,such as cytoskeleton assembly,with the help of GO and KEGG analysis.In a word,the composite had a hierarchical pore structure of micropores and mesoporous with the advantages of simple preparation and strong targeting ability to mercury-binding proteins.The establishment of the analytical method provides an effective research method and means for the screening,identification and protein-protein interaction of mercury metal-binding proteins in membranous nephropathy.It also lays a foundation for further clarifying the effect of heavy metals on membranous nephropathy.