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MALDI mass spectrometry new methods and new technologies and their applications in proteomics

Author: ZhangYing
Tutor: YangPiYuan
School: Fudan University
Course: Analytical Chemistry
Keywords: Proteomics Matrix-assisted laser desorption ionization time-of-flight Target Enrichment Desalination Block polymer Enzymatic Dendrimer Carbon nanotubes Matrix Phosphopeptides Oligosaccharides Phospholipid Non - small cell lung cancer
CLC: Q51
Type: PhD thesis
Year: 2009
Downloads: 1231
Quote: 1
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Abstract

The PhD thesis work is mainly based on matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS), new methods and new technology development and its application in the field of proteomics research. Trace peptide enrichment desalination research has made innovative progress, the use of block copolymers successfully achieved the the sample efficient target on in situ desalination and enrichment directly for MALDI-TOFMS analysis, and successfully applied to the actual samples of mouse liver proteomics research. Directly on the target protein hydrolysis technology, the development of a dendrimer - Carbon Nanotubes as a substrate, trypsin soluble immobilized enzyme is fixed to the surface of the dendrimer, successful implementation of the protein in the target plate The rapid enzymatic directly for MALDI-TOFMS analysis, especially suitable for low-abundance proteins efficient digestion and mass spectrometry. To carry out research work for phosphorylated peptides, oligosaccharides and phospholipids ionization efficiency and low in the matrix, the matrix system, greatly improving the efficiency of the three types of molecular ionization, for high sensitivity mass spectrometry detection solutions. MALDI-TOFMS direct analysis of tissue sections for non-small cell to carry out research work, tissue imaging methods to find biomarkers in non-small cell carcinoma, and found that found in non-small cell lung squamous cell cancer is different from the adjacent tissue found m / z 3000-3500 cluster peak; 2 subtypes of non-small cell lung carcinoma and lung adenocarcinoma compared to the respective characteristic peaks of phospholipid molecules; cancer tissue heme b (m / z 616.2) expression levels much lower than the adjacent tissues, the difference between non-small cell lung cancer and adjacent tissues biological markers. With the completion of the human genome sequence, \Since then, the interpretation of human life \Proteomics (Proteomics) is the life sciences into one of the hallmarks of the genomic era. Proteomics proteome Proteome Research. The proteome originally expressed by the genome of a cell or a tissue, all of the corresponding protein. Determination of the genome of an organism expressed the idea of ??total protein, germination in 1975 by two-dimensional gel electrophoresis of the invention. 1994 Williams formally proposed the term \magazine. Now the concept has been extended, refers to an organism protein and its mode of existence of all gene expression, or is a cell, tissue or whole organisms in a particular time and space have a full set of proteins, and protein expression after modification, protein-protein interactions, the spatial structure of the protein and the more advanced composites. Four basic supporting technology for protein separation techniques, protein identification technology, protein-protein interaction analysis and bioinformatics data processing technology called proteomics. Among them, protein identification, mass spectrometry technology in a variety of methods for identification of proteins of the fastest growing, most widely used and the most development potential. From the the John Finn (JohnB.Ferm) and Tianzhonggengyi (Koichi.Tanaka) invented two soft ionization mode electrospray ionization ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) technology Since the establishment of confirmation and structural analysis of biological macromolecules, mass spectrometry has become one of the most active in the field of cutting-edge research field of life sciences. Scientific research in proteomics was able to achieve rapid development mainly depends on the rapid development of mass spectrometry and separation and analysis of high-throughput technology breakthrough progress. However, due to the variability and diversity of protein, proteome research in analytical techniques and analytical instruments there are many bottleneck issues to be resolved. Such large differences in the level of expression of the protein, and a wide dynamic range, existing separation technologies is not enough to an in-vivo protein separated: the existing analytical sensitivity of the instrument is difficult to trace protein in vivo precise analysis, which kinds of trace proteins play a key role in the process of life. With the further research of proteomics the traditional biological mass spectrometry technology platform has enough to deal with highly sensitive and high-throughput proteomics high accuracy testing requirements, research and development is based on new technologies and new methods of biological mass spectrometry promotion of proteomics research is of great significance. In this thesis, the development of MALDI mass spectrometry analysis of the new techniques and methods as the starting point, to carry out the research work. This thesis is divided into five chapters, the main contents are summarized as follows: The first chapter of proteomics research and development profile, including proteomics research purpose, meaning, the main research methods, and the application of proteomics in the study of human disease . Mass spectrometer as an important technical support in the field of proteomics research, the development of this section of the mass spectrometer's a simple overview of the core components of the ion source, mass analyzer and detector technology development. With a summary of the principles and applications focus on two widely used in proteomics mass spectrometry matrix-assisted laser desorption mass spectrometry and electrospray ionization mass spectrometry. This chapter also summarizes the proteomic analysis of challenges facing analysis of MALDI importance of research and challenges, and on this basis, the research direction of this project work. The second chapter of the research main block copolymer PSF-b-PEO used to the desalting and enrichment of trace peptide target directly analyzed by MALDI-TOFMS. We chose the microscopic phase separation structure of the block copolymer PSF-b-PEO as MALDI target coating, and then use the the produced polymer coating desalting and enrichment of trace polypeptide target. The structure of the block copolymer having hydrophilic and hydrophobic ends, hydrophilic end point overlying the sample solution, due to the hydrophobic end is not dissolved in the sample solvent, maintaining the stability of the block polymer in the metal film; and salts and contaminants having strong adsorption, and thus in the process of sample solvent evaporation, salt and other contaminants are firmly encased within the polymer dissolved in the sample solvent. No longer enter into the point overlying the substrate solution, the salt is wrapped within the polymer, the solvent of the matrix, while the peptide will be dissolved again in the matrix and the matrix-forming crystalline. Further, since the hydrophobicity of the polymer film so that the sample will shrink in a small target area so that the sample has been enriched on a target. Therefore no additional cleaning desalination, the sample enrichment desalination direct MALDI-TOFMS analysis. Chapter research work mainly dendrimer - carbon nanotube substrate, trypsin soluble immobilized enzyme fixed on the surface of the dendrimer, successful implementation of trace amounts of protein in the target plate rapid enzyme solution for MALDI-TOFMS analysis. First of carbon nanotubes in the mixed acid for acidification in the surface of the truncated carbon nanotubes by acidification and the formation of a large number of carboxyl groups and to remove impurities. And then combined in the polyamide amine - type dendritic polymers (dendrimers) by covalent binding to the surface of carbon nanotubes, and trypsin fixed in the amino terminus of the polymer by glutaraldehyde crosslinking reaction. Carbon nanotubes surface-modified dendrimers, which greatly enhances the solubility of carbon nanotubes. Accordingly, the immobilized enzyme substrate to the dendrimer - carbon nanotube having the advantages of high stability of the immobilized enzyme has the advantage of high soluble enzyme activity, and does not need to be due to its solubility, hydrolysis after carbon nanotubes are separated from the solution can be identified by mass spectrometry. Dendrimer - carbon nanotubes the matrix immobilized trypsin enzymatic target, high efficiency, speed, enzyme from degradation peak, after the completion of the hydrolysis can be directly detected by mass spectrometry, particularly suitable for trace protein digestion and identification. The fourth chapter of the research work for improving the MALDI matrix system of phosphorylated peptides, oligosaccharides and phospholipids ionization efficiency carried out. The ammonium salt is added to the substrate solution to improve the ionization efficiency of the phosphopeptide; Synthesis of novel ionic matrix of 2,3,4 - Trihydroxyacetophenone / N, N-dimethylaniline (2,3,4 - THAP / DMA), 2,3,4 - Trihydroxyacetophenone / pyridine (2,3,4-THAP / Py), of 2,4,6 - Trihydroxyacetophenone / N, N-di-methylaniline (2,4,6-THAP / DMA) and 2,4,6 - Trihydroxyacetophenone / pyridine (2,4,6-THAP / Py) improve the ionization efficiency of the oligosaccharide; addition, extension of the non- polar matrix [2E-3-(4 - tert - butylphenyl) -2 - methylpropan-2 - the alkenylene yl] malononitrile (DCTB), application range, find its ionization efficiency in improving the phospholipid aspect has an important contribution. Greatly improving the ionization efficiency of the three types of molecules, high sensitivity mass spectrometry detection solutions work for MALDI mass spectrometry imaging technology for the analysis of non-small cell lung cancer tissue sections. MALDI mass spectrometry imaging technique for direct analysis of tissue sections has been rapid development in basic and clinical research. The organization of molecules such as proteins, polypeptides, such distribution information by MS scan of the surface of the frozen tissue sections can be quickly and intuitively. We use mass spectrometry imaging technology on human non-small cell lung cancer tissue sections and adjacent tissue sections were studied, MS direct tissue surface analysis method has been optimized, the initial establishment of biological imaging mass spectrometry method to find non-small cell lung cancer Method markers. The results showed that lung squamous cell carcinoma the characteristic cluster peaks within the scope of the m/z3000-3500; non-small cell subtypes of lung squamous cell carcinoma and lung adenocarcinoma compared to the respective characteristic peaks of phospholipid molecules ; and heme b expression in lung cancer is much lower than adjacent tissues. Mass spectrometry for direct tissue section analysis technology to visually reflect the difference between cancer and normal tissues at the molecular level, is expected to further improve the accuracy of clinical diagnosis of lung cancer.

MALDI mass spectrometry new methods and new technologies and their applications in proteomics

Abstract

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