Xiang, Fan (1997) Improvement and investigation of sample preparation for matrix-assisted laser desorption/ionization of proteins. Doctoral (PhD) thesis, Memorial University of Newfoundland.
- Accepted Version
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Since the introduction of Matrix-Assisted Laser Desorption and Ionization (MALDI) in 1988 the application of MALDI mass spectrometry has become a vital research field. The method of analyte/matrix preparation plays a central role for achieving optimal performance in MALDI. -- In the first part of this work, a new method of preparing the samples to increase contaminant tolerance is described. While the solvent composition and matrices used in the "dried droplet" method have changed since MALDI was introduced, there has been little fundamental change in this protocol. The "dried droplet" method for MALDI MS is a simple one step process, but high concentrations of nonvolatile liquid, such as glycerol, urea, DMSO and other protein stabilizers, can reduce or eliminate the intensity of protein ion signals. Instead of using the one step process, we developed a three step process. The examples presented in this part of this thesis illustrate how analyses were improved as a result of the new sample preparation procedure. The sample films produced by the new method adhere firmly to the substrate, allowing easier washing of the film compared to the dried-droplet deposits. The films were more uniform than dried-droplet deposits with respect to ion production. The presence of non-proteinaceous material at high concentrations in the solution did not seriously affect ion production from these films. -- The basic difference between laser desorption/ionization and matrix-assisted laser desorption/ionization is that the laser radiation is directly absorbed by the analyte in the former case, while the matrix dominates energy absorption and the formation of ions in the latter. In the second part of this work, the influence of matrix concentration on analyte ion yield and the mass resolution of the molecular ion signals were investigated. MALDI time-of-flight mass spectra of protein mixed at different concentrations in each of gentisic acid, trans-α-cyano-4-hydroxycinnamic acid and sinapic acid matrices, were obtained. The results showed that the intensities of the analyte ion peaks increase gradually with an increase in concentration of matrix in solution and then decrease slightly as the matrix concentration continues to increase. The analysis of the spectra also showed that the molecular ion peaks are broadened, and the mass resolution decreases, as the matrix concentration increases. A proposed interpretation of these results is given based on two categories of matrix concentration effects: a physical effect involving energy deposition and redistribution processes, and a chemical effect involving co-crystallization of the matrix and analyte followed by photochemical ionization. -- Poor crystallization of a MALDI sample has a negative effect on its ability to generate ions. In the last part of this work a strategy was demonstrated to alleviate this problem. Certain amounts of impurities (or additives), present in solution during crystallization, have a pronounced effect on the crystallization process, in particular on the morphology of the deposited crystals. The technique of controlling crystal growth by additives was also discussed. The crystallization of gentisic acid mixed with its isomers and other benzoic acid derivatives was then investigated. The performance of the resulting crystals in MALDI MS was evaluated. The results showed that the admixture of gentisic acid with 2,3-dihydroxybenzoic acid or 3-amino-2,5,6-trifluorobenzoic acid homogenized the crystal morphology, and consequently improved the MALDI MS performance.
|Item Type:||Thesis (Doctoral (PhD))|
|Additional Information:||Bibliography: leaves 104-113.|
|Department(s):||Science, Faculty of > Physics and Physical Oceanography|
|Library of Congress Subject Heading:||Laser spectroscopy; Proteins--Analysis--Methodology; Proteins--Spectra; Matrix isolation spectroscopy|
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