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Dissertation
Exploration of the quantitative aspects of matrix-assisted laser desorption ionization time-of-flight mass spectrometry
Doctor of Philosophy (Ph.D.), Drexel University
Jan 2013
DOI:
https://doi.org/10.17918/etd-4066
Abstract
This thesis focuses on the study of different methods for preparing samples for analysis by MALDI TOFMS and application of these methods to quantify proteins of large size, including insulin, cytochrome c and myoglobin. Methodology for effective ion-exchange of the matrix, solvent and analyte was developed to improve the MALDI spectrum by reducing the amount of alkali adduction observed. Measurement of the alkali metal contamination using atomic absorption spectroscopy shows a significant decrease in concentration by comparing materials before and after ion-exchange. Recrystallization and some preliminary studies using spincolumn desalting for the analyte were also investigated. The electrospray deposition technique was initially employed to optimize its operating parameters. A two-level plus center point factorial design experiment was performed on three device parameters. Several statistically significant factors were identified using several experimental responses, including the absolute peak area and the relative standard deviation of the peak area from replicate measurements. However, when the optimized conditions were used in the analysis of a higher molecular weight protein, it was discovered that there were problems with the use of a high percentage of organic solvent in the sample preparation. To overcome this limitation the oscillating capillary nebulizer (OCN) was investigated as an alternate means of depositing the sample now prepared in a high water content solution. As the OCN can handle 100% aqueous solutions and produce a homogeneous sample surface, this sample deposition technique was chosen for further study. The device was custom fabricated and factorial-design experiments were employed to optimize the OCN parameters. Several statistically significant factors were identified and the optimized conditions were then used in quantitative studies of larger proteins. A vacuum stability study on the prepared MALDI samples as well as a heating study was also conducted. The optimized OCN device was used to prepare cytochrome c samples for quantitative analysis over the range of 4-10 nmole/mL; the work included determination of the detection limit of the method. The OCN was also used to study the matrix-to analyte ratio requirements for myoglobin with the matrix 2,5-dihydroxybenzoic acid (DHB) and cytochrome c with the matrices DHB and 6-aza-2-thiothiamine (ATT).
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Details
- Title
- Exploration of the quantitative aspects of matrix-assisted laser desorption ionization time-of-flight mass spectrometry
- Creators
- Runbo Li - 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
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Arts and Sciences; Chemistry; Drexel University
- Other Identifier
- 4066; 991014632670204721