Dissertation
Development of a cell free MALDI TOFMS method for the identification of Saccharomyces cerevisiae yeast strain & studies of the molecular properties of MALDI matrices
Doctor of Philosophy (Ph.D.), Drexel University
May 2018
DOI:
https://doi.org/10.17918/D8VQ25
Abstract
This thesis is divided into two parts, where the molecular properties and salt contamination of MALDI matrices are first investigated, followed by the development of a cell-free method for the identification of yeast strains by MALDI TOFMS. In Part 1, the solubility of several common MALDI matrices are measured in a variety of solvents and mixtures of acetonitrile and water. In addition to the solubility measurements, sublimation and recrystallization methods are used to reduce the alkali contamination present in commercially available MALDI matrices. Work in both areas resulted in improved sample preparation, which aided the work described in part 2. In Part 2 of the thesis, a cell-free method was developed as an alternative to the currently used Bruker Biotyper method for the identification of Saccharomyces cerevisiae yeast strains via MALDI TOFMS analysis. SPE was used for concentrating the desired peptide/protein analytes and for removing interferences. Factorial design optimization experiments were performed to increase the efficiency of the SPE method, while MALDI TOFMS in combination with the Bradford protein assay were used to monitor the optimization. Problems were encountered with the Bradford assay, including interference from the presence of acetonitrile in the elution solution. Results showed that the extent of interference depends on the protein used as the standard for the assay; Angiotensin I was found to be a better standard than BSA, and relationship between peptide standard size and the number of dye molecules bound to the protein was uncovered. Yeast strains were successfully identified using the Bruker Biotyper method which requires the lysing of viable cells to obtain signal from ribosomal proteins. As an alternative, a new cell-free approach is introduced where liquid samples of the cell supernatant is used for the analysis. The new approach is also successful in correctly identifying yeast strains, and it demonstrated 33% success rate in identifying the yeast strain used to brew 3 homebrewed beers.
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Details
- Title
- Development of a cell free MALDI TOFMS method for the identification of Saccharomyces cerevisiae yeast strain & studies of the molecular properties of MALDI matrices
- Creators
- Elsa Gorre - DU
- Contributors
- Kevin Glenn Owens (Advisor) - Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xxiii, 310 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Arts and Sciences; Chemistry; Drexel University
- Other Identifier
- 8105; 991014632384604721