Investigations On Protein Enrichment And Activity Assay With Mass Spectrometry

Proteins are functional implementers of living systems.Their activities depend on the states of themselves.Of all the ways that regulate protein activities,post-translational modification is the most common one that widely exists in life and plays important roles in a variety of biological processes,including material transportation,gene replication,cell proliferation,differentiation and apoptosis.Therefore,the study of protein post translational modifications,as well as the analysis of protein activities,will help to further understand protein structures and functions,uncover the relationship between diseases and protein activities,and develop clinical diagnosis and treatment protocols of related diseases.This dissertation combines mass spectrometry(MS)with the technologies of nanomaterials,coding,microarray,and mass spectrometric imaging to design a new functional material for rapid separation and selective enrichment of glycopeptides,and develop a series of methods for multiple enzyme acti’vity assay.It contains the following five parts:1.Mercaptophenylboronic acid modified gold nanoparticle@silica bubbles for buoyant separation and specific enrichment of glycopeptidesA buoyant separation method was proposed using mercaptophenylboronic acid modified gold nanoparticles@silica bubbles(MPB-Au＠SiBs),a new functional low-density material,for specific enrichment of glycopeptides.The step-by-step synthesis of MPB-Au@SiBs was simple and mild.Compared with conventional boronic acid-functionalized materials,the introduction of buoyant substrate avoided high-speed centrifugations or the use of external magnets.Based on the recognition of boronic acid to the cis-diol OH-containing structures in most sugar moieties,the material could specifically capature glycopeptides,and subsequently release them by changing solution acidity for matrix assisted laser desorption/ionization time-of-flight(MALDI-TOF)MS analysis.The newly prepared MPB-Au@SiBs showed great performance in enrichment capacity,sensitivity and selectivity,and simplified the separations of glycopeptides from biological samples,thus possessed potential applications in glycoproteome analysis.2.Peptide code-on-a-microplate for protease activity analysis via MALDI-TOF mass spectrometric quantitationA peptide-encoded microplate was proposed for MALDI-TOF MS analysis of protease activity.The peptide code was designed to contain a coding region and the substrate of protease for enzymatic cleavage.Upon the cleavage reaction in the presence of target protease,the coding region was released from the microplate,which was directly quantitated by using a peptide with one-amino acid difference as the internal standard.The m/z value of the coding region could be used as the unique"Protease ID" for its identification,and the amount of the cleaved peptide sequence could be used for protease activity analysis.As a concept proof of multiple protease assay,a microplate was prepared with two peptide codes containing different coding regions and substrates for respective trypsin and chymotrypsin,which were used as model proteases to demonstrate the feasibility.The microplate showed outstanding selectivity.It provided a powerful tool for convenient identification and activity analysis of multiple proteases.3.Peptide codes for multiple protease activity assay via high-resolution mass spectrometric quantitationIn order to overcome the disadvantages of quantitation with MALDI-MS,a peptide coding strategy for label-free analysis of multiple protease activities was proposed using high-resolution mass spectrometry(HRMS).The method was validated by employing trypsin and chymotrypsin as models.Quantitative curves could be obtained by plotting the peak areas of’ "Protease IDs" vs trypsin and chymotrypsin concentrations,respectively.Compared with previously proposed method,this platform showed higher sensitivities and could realize accurate quantitations without aid of internal standards.It was of great potential in the applications of clinical diagnosis.4.An enzyme assay strategy with enhanced confidence by the combination of selenium-containing peptides and high-resolution mass spectrometryIn order to simplify the identification of enzymatic products in complex samples and obtain credible data for quantitative analysis,a selenium-containing peptides-high resolution mass spectrometric method was developed for comprehensive study of enzyme activities.After introducing a selenomethionine into each sequence of peptides and treating the peptides with bio-samples,corresponding products and substrates could be rapidly distinguished and identified by the feature of peculiar Se-isotopic distributions in the full-scan mass spectrum.The capacity of the proposed method to exclude tiny-mass-different interferences was also demonstrated by matching experimental ratios of selected isotopic peak areas with theoretical values to examine the authenticity of data and to perform reliable quantitation.This proposed method was highly promising for high-throughput enzyme assays and could be extended to studies of the synergistic effect of multi-enzymes and substrate screening.5.MALDI-MS patterning of caspase activities and its application in the assessment of drug resistanceTo achieve high throughput of enzyme activity assay,a MALDI-MS patterning strategy for convenient visual presentation of multiple enzyme activities was developed with an easy-to-prepare chip.The array-based caspase activity pattern chip(Casp-PC)was fabricated by assembling different phospholipid-tagged peptide substrates on a modified ITO slide.The advantages of amphipathic phospholipid enhanced the bio-campatibility of the chip and assured high-quality mass spectrum for imaging analysis.Upon the respective cleavage of these substrates by different caspases,such as caspase-1,-2,-3,and-8,to produce a mass shift,the enzyme activities could be directly evaluated by MALDI-MS patterning with m/z-dependent coding image of the cleavage products.The abilities of this strategy to identify drug sensitive/resistant cancer cells and assess curative effects of anticancer drugs were also demonstrated,and could be easily expanded to the analysis of other enzyme systems,indicating the promising applications of the method and the designed chip.