Study On Novel Mass Spectrometic Methods For Analysis Of Peptides And Small Biomolecules

Since the introduction of two soft ionization methods,named as matrix-assisted laser desorption/ionization(MALDI)and electrospray ionization,mass spectrometry(MS)has been become one of the most important characterization tools for bioanalysis.MALDI-TOF MS has been a powerful tool in the modern life sciences due to its high throughput,low detection limit,mixtures analysis ability as well as good tolerance to sample contamination.In biological study,the involved real-world samples are complex,while disease associated biomolecules that are known as biomarkers expresses commonly at low abundance.Therefore,it is necessary to develop new techniques and methodologies for the rapid and effective sample analysis based on MS.In this dissertation,by combining nanotechnology,surface functionalization,chemical derivatization and chemical biology with traditional bioanalytical methods,a series of newly-designed bioanalytical methods to resolve existing problems in specific enrichment and mass spectrometric detection biomolecules has been developed.This dissertation includes the following four parts:1.Surface-assisted laser desorption/ionization mass spectrometric detection of biomolecules using functional single-walled carbon nanohorns as matrixA surface-assisted laser desorption/ionization time-of-flight mass spectrometric(SALDI-TOF MS)method was developed for analysis of small biomolecules by using functional single-walled carbon nanohoms(SWNHs)as matrix.The functional SWNHs could transfer energy to the analyte under laser irradiation for accelerating its desorption and ionization,which led to low matrix effect,avoided fragmentation of analyte and provided high salt tolerance.Biomolecules including amino acids,peptides and fatty acids could successfully be analyzed with about 3-and 5-fold higher signals than those obtained using conventional matrix.By integrating the advantages of SWNHs and the recognition ability of aptamer,a selective approach was proposed for simultaneous capture,enrichment,ionization and MS detection of adenosine triphosphate(ATP).This method showed greatly improved detection limit down to 1.0 ?M for analysis of ATP in complex biological samples.This newly designed protocol not only opened a new application of SWNHs,but also offered a new technique for selective MS analysis of biomolecules based on aptamer recognition systems.2.Highly selective enrichment of phosphopeptides with high-index facets exposed octahedral tin dioxide nanoparticles for mass spectrometric analysisHigh-index facets exposed octahedral tin dioxide(SnO2)nanoparticles were successfully synthesized and applied to selectively enrich phosphopeptides for mass spectrometric analysis.The high selectivity and capacity of the octahedral SnO2 nanoparticles were demonstrated by effectively enriching phosphopeptides from digests of phosphoprotein(?-or ?-casein),protein mixtures of ?-casein and bovine serum albumin,milk,and human serum samples.The unique octahedral SnO2 with abundant unsaturated coordination Sn atoms exhibited enhanced affinity and selective coordination ability with phosphopeptides due to their high chemical activity.The strong affinity led to highly selective capture and enrichment of phosphopeptides for sensitive detection through the bidentate bonds formed between surface atoms and phosphate.The phosphopeptides could be detected in p-casein down to 4×10-9 M or in the mixture of ?-casein and BSA with a molar ratio of even 1:100.The performance in selective enrichment of phosphopeptides from drinking milk and human serum showed powerful evidence of high selectivity and efficiency in identifying thelow-abundant phosphopeptides from complicated biological samples.This work provided a way to improve the physical and chemical properties of materials by tailoring their exposed facets for selective enrichment of phosphopeptides.3.Facile synthesis of boronic acid-functionalized magnetic carbon nanotubes for highly specific enrichment of glycopeptidesA stepwise strategy was developed to facilely synthesize boronic acid functionalized magnetic carbon nanotubes(MCNTs)for highly specific enrichment of glycopeptides.The MCNTs were synthesized by a solvothermal reaction of Fe3+loaded on the acid-treated CNTs and modified with 1-pyrenebutanoic acid N-hydroxysuccinimidyl ester(PASE)to bind aminophenylboronic acid(APBA)via an amide reaction.The introduction of PASE could bridge the MCNT and APBA,suppress the nonspecific adsorption and reduce the steric hindrance among the bound molecules.Due to the excellent structure of the MCNTs,the functionalization of PASE and then APBA on MCNTs was quite simple,specific and effective.The glycopeptides enrichment and separation with a magnetic field could be achieved by their reversible covalent binding with boronic group of APBA-MCNTs.The exceptionally large specific surface area and the high density of boronic acid groups of APBA-MCNTs resulted in rapid and highly efficient enrichment of glycopeptides,even in the presence of large amounts of interfering nonglycopeptides.The functional MCNTs possessed high selectivity for enrichment of 21 glycopeptides from the digest of horseradish peroxidase,which was demonstrated by MALDI-TOF mass spectrometric analysis to show more detectable number of glycopeptides than 9 glycopeptides with commercially available APBA-agarose.The proposed system also showed better specificity to glycopeptides even in the presence of non-glycopeptides with 50 times higher concentration.The boronic acid functionalized MCNTs provide a promising selective enrichment platform for precise glycoproteomic analysis.4.A dual quinone tagging strategy for tagging and quantitative detection of cysteine-containing peptidesSelective tagging of specific amino acids is an interesting protocol in protein profiling by peptide mass fingerprinting.In this work,a facile selective method for tagging cysteine-containing peptides(Cpep)has been proposed using electrophiles substituted benzoquinones,which achieves high specificity and sensitivity.With this strategy,we can identify the unique peptides from a complex peptide mixture as well as count the number of cysteine residues through the direct observation of untagged and tagged products.Such a tagging strategy is simple and does not cause sample loss,providing powerful information to facilitate accurate and comprehensive protein identification.Taking the advantages of the efficient tagging ability of benzoquinones for cysteine-containing peptides,a dual quinone tagging method is proposed for MALDI quantitative detection of cysteine-containing peptides.Benzoquinone tagged Cpep that differs in only one methyl from the signal peptide(methyl-p-benzoquinone tagged Cpep)is added as internal standard.The ratio of signal intensity of the signal peptide to the internal standard can be plotted against the concentration of Cpep to show a linear relation within a concentration range from 5 nM to 5000 nM.The limit of detection is as low as 2 nM(0.5 fmol).Because of the simplicity,high-speed,sensitiveness,and versatility of the propose strategy,we believe it has a bright future in the large-scale detection and analysis of proteins.