By Maldi Mass Spectrometry Of New Methods And New Technology And Its Application In Proteomics Research

The main contribution of this doctoral dissertation is the development of new methods and techniques of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and their application for proteomics research. Carrying out discoveries and inventions of the enrichment and desalting of low-abundance peptides using block copolymer and combined with MALDI-TOFMS analysis, which were then successfully applied in real sample analysis; Development of novel on-plate high speed proteolysis method with water-soluble immobilized trypsin on dendrimers-modified-carbonnanotubes (dCNTs) followed with MALDI-TOFMS analysis, which is especially helpful for low-abundance proteins digestion and identification; Investigation of new matrices system for high ionization of molecules includes phosphopeptides, oligosaccharides and phosphatidylcholines, their ionization efficiency were in a large extent enhanced with these novel matrices systems; Using MALDI-TOFMS for directly tissue section profiling analysis of non-small cell lung cancer(NSCLC). Results indicated that cluster peaks appears in the m/z ranges from 3000 to 3500 corresponding to squamous cell carcinoma tissues; The phospholipids profiles from different subtypes of NSCLC were investigated, and there were obvious differences between squamous cell carcinoma tissues and adenocarcinoma tissues; Also, results showed m/z 616.2 corresponding to heme b showed low ion intensity in cancerous regions compared with paracancerous.As the human genome sequence "encyclopedia" completed, it ushered a new epoch of deciphering the mystery of life and afforded "anatomic map"of human life at the molecular level for the first time. It fueled the deciphering of "life codes", and made the genomics entering the post-genomics era since then. "Proteomics", as a new frontier in life science, has become one of the landmarks for the post-genomics period. Proteomics studied with the proteome as its research object. The term "proteome" was first coined to describe "the set of proteins encoded by the genome from a given cell or tissue". Determination of all the proteins from an organism was firstly germinated in 1975 at the time invention of two-dimensional gel electrophoresis. In 1994, Willams proposed this question formally, but the term "Proteome" was created by Wilkins, it comes from the term "Protein" and the term "genome". Nowadays, the concept of "Proteome" is expanded, it refers to not only all the proteins in any given cell, but also the set of all protein isoforms and modifications, the interactions between them, the structural description of proteins and their higher-order complexes etc.Protein separation, protein identification, protein interaction analysis and bioinformatics data processing are four basic support techniques in proteomic study. As for protein identification, mass spectrometry technology is the fastest developing, the most extensive application and the most promising technology in the various protein identification methods currently. From the invention of two important soft ionization methods "electrospray ionization"(ESI) and "matrix-assisted laser desorption ionization"(MALDI) technology by JohnB.Fenn and Koichi.Tanaka respectively, as well as the establishment of biological macromolecules identification and structure analysis method, mass spectrometry(MS) has now become the most active frontier research field in life sciences. The vigorously development of proteomics mainly rely on development of mass spectrometry technology and progress of rapid protein separation and analysis technique with high-throughput. However, because of the diversity and variability of protein, proteomics studies are far more complex and much more difficult than genomics studies, especially bottlenecks will arise in analytical techniques and instruments. For example, the separation capability of existing technology is not enough to isolate all of the proteins in a given biological because of the wide dynamic range of the protein expression level; the sensitivity of the existing analysis instrument is hard to obtain traces of proteins, even though the precise analysis of trace protein plays an important role in life process. With the further in-depth study of proteomics, traditional biomass spectrometry platform is not enough to cope with detection requirements with high sensitivity, high accuracy, and high throughput. Therefore, the development of new methods and new techniques of biomass spectrometry is of great significance for promoting proteomics research.This thesis focused on the development of new methods and new techniques of mass spectrometry analysis. The whole doctoral dissertation consists of five chapters and the contents are summarized as follows:The first chapter summarized the development situation of proteomics research, including its research purpose, scientific significance, main research methods, and its application in human diseases research. As an important technical support platform of proteomics research fields, the development of mass spectrometry instrument, including its core components like ion source, quality analyzer and detector technology were reviewed in this chapter. Especially, it focused on the introduction of the principle and application of the most two widely used mass spectrometry (MALDI-MS and ESI-MS) in proteomics. At last, we introduced the challenges that proteomics analysis still faced. Based on that, the investigation direction and purpose of this dissertation was put forward.The research work describe in chapter two focused on describing a novel method of on-plate desalting and enriching of trace peptides with block copolymer PSF-b-PEO and combined with MALDI-TOFMS analysis. We choose the block copolymer PSF-b-PEO with microscopic phase separation structure as the MALDI target coating materials, and then made use of the polymer film for on-plate desalting and enriching of trace peptides. There are hydrophilic and hydrophobic domains in its structure, after the sample solution added on the polymer film, because hydrophobic domain could maintain the polymer do not dissolve in the sample solution thus keeping the stability of the polymer film. The hydrophilic domains could dissolve in samples solutions and had strong effect on adsorption of salts or other contaminates, thus with the evaporation of sample solvent, salts and other contaminates were firmly wrapped inside the polymer. After added the matrix solution, salts no longer entered into the matrix solution due to the adsorption of polymer, whereas peptides redissolved in the matrix solution and form crystals with the matrix. In addition, the sample shrunk in a small target area on the hydrophobic surface of the polymer film, thereby, the sample was enriched on-target. Therefore, after the simultaneous desalting and enriching process, the sample could be directly analyzed by MALDI-TOFMS without any additional washing process.The research work describe in chapter three focused on describing a novel method on-plate proteolysis with immobilized trypsin on dendrimers-modified-carbon nanotubes followed with MALDI-TOFMS analysis. Firstly, we treated the carbon nanotubes in mixed acid reagent to form large amount carboxyl group on its surface, and cut short of the carbon nanotubes as well as removed impurities through the acidification treatment. Then the polyamide-amine dendrimers was covalent modified on the carbon nanotubes surface. After that, trypsin was immobilized onto the dendrimers-modified carbon nanotubes(dCNTs) through the reaction of the aldehyde groups with the amine groups on the end of dendrimers. The dendrimers modification in a large extent enhanced the water-solubility of the carbon nanotubes. Therefore, the trypsin-linked dCNTs represents innovative properties that possess the advantages of both good stability of immobilized enzymes and high activity of soluble enzymes. Moreover, because of its good water-solubility, after the completion of proteolysis it can be used for directly mass spectrometric analysis without any need to remove the carbon nanotubes. It afforded many benefits using the trypsin-linked dCNTs for on-plate proteolysis, such as high digestion efficiency, high digestion speed, less enzymatic autolysis and can be directly used for MS analysis after the completion of digestion, making it especially suitable for proteolysis and identification of trace protein.The research work describe in chapter four focused on describing some novel matrices systems for enhanced the ionization of phosphopeptides, oligosaccharides or phosphatidylcholines. Added ammonium phosphate salts in matrix solution to improve the ionization efficiency of phosphopeptide; Synthesized novel ionic matrices 2,3,4-THAP/DMA, 2,3,4-THAP/Py, 2,4,6-THAP/DMA and 2,4,6-THAP/Py to improve the ionization efficiency of oligosaccharides; Expanded the application of nonpolar matrix T-2-(3-(4-t-Butyl-phenyl)-2-methyl-2-propenylidene)malononitrile(DCTB) to improve the ionization efficiency of phosphatidylcholines.The research work describe in chapter five described the method using MALD1-T0FMS for directly profiling of non-small cell lung cancer(NSCLC) tissues. MALDI-MS profiling for tissue section analysis is rapidly developed among foundation and clinical medical research. Through scanning on the frozen tissue slices, distribution information of molecules such as protein and peptides from a certain tissue are directly obtained in little time by Profiling MS technology. Based on the Profiling MS technology, human being's NSCLC tissues and paracancerous tissue slices were investigated, a profiling MS method analysis of NSCLC for biomarkers has been established after optimization the related methods. Results indicated that cluster peaks appears in the m/z ranges from 3000 to 3500 corresponding to corresponding to squamous cell carcinoma tissues; The phospholipids profiles from different subtypes of NSCLC were investigated, and there were obvious differences between squamous cell carcinoma tissues and adenocarcinoma tissues; Also, result showed m/z 616.2 corresponding to heme b showed low ion intensity in a cancerous regions compared with paracancerous. The profiling MS technique can present the differences between cancerous with paracancerous areas at molecular level, which is hoped to help improving the clinical diagnosis accuracy of NSCLC.