Matrix-assisted Laser Desorption Ionization And Its Application In Proteomics

In this thesis,the Matrix Assisted Laser Desorption Ionization(MALDI) is studied. Based on the photo-electro principle of the MALDI process,a number of in-source redox reactions have been developed by using a semiconductor TiO₂ substrate as the target plate.These in-source reactions are further employed for the purpose of proteome research.Besides,in-situ enrichment of phosphopeptides has been realized on the TiO₂ modified MALDI target plate.Proteolysis in nanopores is also studied for accelerating the digestion reaction rate.In chapter 1,recent research progress on bio-mass spectrometry and proteomics is described.These progresses include the study on the principle of MALDI process, inorganic material assisted laser desorption ionization,the photo-electro character of semiconductor nano-TiO₂,mass spectrometer based sample fragmentation,peptide tagging technique,protein post translational modification and immobilized protease for fast proteolysis.Chapter 2,3 and 4 are the research parts of the thesis.In chapter 2,based on the photo-electro principle of the laser desorption ionization(LDI),many in-source redox reactions have been developed by using the semiconductor TiO₂.These in-source reactions have been employed for amino acid on-line tagging,disulfide on-line cleavage and peptide on-line oxidative fragmentation.In chapter 3,for analyzing the protein post translational modifications,in-situ enrichment and detection of phosphopeptides has been performed on the TiO₂ modified target plate.In Chapter 4, in-nanopore digestion of proteins has been studied for fast proteolysis.The detailed points of this thesis are expended as follows:1.In-source redox reactions during the laser desorption ionization.●MALDI in-source photo-oxidation reactions for on-line peptide taggingHere,we use the addition reaction of oxidized hydroquinone on cysteine containing peptides.Upon irradiation by the UV laser,hydroquinone is photo-oxidized to benzoquinone or other active intermediates that can in turn react with cysteine containing peptides to tag them concomitantly to the sample ionization.Indeed, selective tagging of specific amino acids is an interesting protocol in protein profiling by peptide mass fingerprinting.In this case,the unique peptides can be revealed from the digested proteomes,providing powerful information to facilitate the accurate and comprehensive protein identification.To illustrate this application,the in-source reaction was employed to specifically recognize cysteine-containing peptides from the complex tryptic digests ofβ-lactoglobulin A and albumin,respectively.●In-source photocatalytic reduction of disulfide bond during laser desorption ionizationAs a matter of fact,photocatalytic reductions are much less frequently studied than oxidations,probably because the reducing ability of a conduction band electron is considerably lower than the oxidizing one of a valence band hole.As reported,the trapping of conduction band electrons on the nanoparticles is very fast.To prevent this kind of electron quenching from on-surface or in-volume recombination,it is anticipated that the addition of an electron donor can theoretically be beneficial by scavenging valence band holes and thereby freeing more reductive equivalents.By adding a electron donor of glucose,this photo-reduction concept could be used to carry out in-source disulfide bond cleavage,which are typical reduction processes employed in proteomics.Indeed,disulfide bond formation is a post-translational modification that strongly stabilizes the 3D structure of proteins,and their cleavages is necessary for the rapid sequencing of proteins containing such modifications.The traditional disulfide profiling approaches require several complex pre-treatment strategies such as digestion,chemical reduction,modification and fractionation. Recently,Tanaka et al.reported a new matrix,1,5-Diaminonaphthalene,to help disulfide reduction for on-line disulfide mapping.Herein,an alternative protocol based on a photo-catalytic reaction using a TiO₂-modified plate is developed to realize the in-source cleavage of disulfide bonds as well as laser desorption ionization with the assistance of hole scavengers.To illustrate the present photo-reductive protocol, human insulin was selected as an example to carry out in-source reduction of disulfide bonds during laser desorption ionization.●Photocatalytic Redox Reactions for In-Source Peptide FragmentationIn source photocatalytic redox reactions based on a photosensitive target plate have been developed to realize peptide fragmentation during laser desorption ionization. Sample peptides and glucose are simply deposited on a spot of sintered TiO₂ nanoparticles.With the irradiation of UV laser on TiO₂,electrons are excited from the valence band to the conduction band,leaving oxidative holes and reductive electrons to drive various in-source redox reactions.Glucose,working here as a hole scavenger and conductor,can favor both on-surface reduction and long distance in-plume oxidation,therefore inducing peptide fragmentation.Intense novel C_α-C backbone cleavage was observed to generate a,x-decay,while the N-C_αbond cleavage was also sometimes obtained to induce c,z-fragmentation but rather weaker.The former dissociation is believed to originate from oxidative routes induced by the valence band holes,based on the oxidation of nitrogen atom at the peptide backbone, including H radical abstraction and electron transfer.In contrast,the latter dissociation is supposed to result from reductive processes by the conduction band electrons, which are then rather similar to electron capture dissociation in tandem mass spectrometry.2.Specific on-plate enrichment of phosphorylated peptides for direct MALDI-TOF MS analysisAn on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation.An array of sintered TiO₂ nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions.These spots were used to selectively capture phosphorylated peptides from peptide mixtures,and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the non-phosphorylated peptides.Beta-casein and protein mixtures were employed as model samples to investigate the selection efficiency.In this strategy,the steps of phosphopeptide capture,purification and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate,which greatly reduces sample loss and simplifies analytical procedures.The low detection limit,small sample size and rapid selective entrapment show that this on-plate strategy is promising for on-line enrichment of phosphopeptides,which is essential for the analysis of minute amount of samples in high throughput proteome research.Furthermore,arrays of TiO₂ nanoparticle spots are coated on the foil either by screen-printing or rotogravure-printing followed by sintering to form a mesoporous layer spot to act as anchor for sample deposition.The single-use TiO₂-Al foils are cheap to produce,easy to use,and well suited for high-throughput proteomics research.3.A nanopore-based reactor for efficient proteolysisA nanoreactor based on mesoporous silicates is described for efficient tryptic digestion of proteins within the mesochannels.Cyano-functionalised mesoporous silicate(CNS),with an average pore diameter of 18 nm,is a good support for trypsin, with rapid in situ digestion of the model proteins,cytochrome c and myoglobin.The generated peptides were analyzed by MALDI-TOF MS.Proteolysis by trypsin-CNS is much more efficient in comparison to in-solution digestion,which can be attributed to nanoscopic confinement and concentration enrichment of the substrate within the mesopores.Proteins at concentrations of 2 ngμL^-1 were successfully identified after 20 min digestion.A biological complex sample extracted from the cytoplasm of human liver tissue was digested using the CNS-based reactor.Coupled with reverse phase HPLC and MALDI-TOF MS/MS,165 proteins were identified after standard protein data-searching.This nanoreactor combines the advantages of short digestion time with retention of enzymatic activity,providing a promising way in advancing the development of proteomics.