Optimization methods for analytical separations: MALDI, dynamic IEF and bioassay guided fractionation

Matrix-assisted laser desorption/ionization (MALDI) is currently a widely-used soft ionization method for mass spectrometric analysis. MALDI has advantages that include low sample consumption, ease of sample preparation, short analysis time and soft ionization. MALDI ionization relies on a matrix that is first ionized and then transfers part of its charge to the analytes. However, each individual matrix works best for a certain molecular weight range of analyte. In the first project, three commonly used matrices are combined to expand the molecular weight range MALDI that can measure in one test. Each of the four factors (three matrices and sample) was tested at five different concentration levels. When combining four factors and five levels of each factor, all the possible combinations would be 45 = 1024. Factorial design dramatically reduced the total runs required and predicted the possible combinations for best results.;Over the past several years, there has been a rapidly increasing interest in proteomics due to its potential for understanding biological functions. The complex nature of the proteome requires multiple separation and analysis methods to be used prior to protein identification. Individual separation methods lack the necessary peak capacity alone and multi-dimensional methods have difficulty in interfacing different methods efficiently. In the second project, a new protein separation technique-dynamic isolelectric focusing (IEF)-was developed. A new theory suitable for dynamic IEF was developed as well. Dynamic IEF has a peak capacity of at least 1100 and is easily connected to other analysis methods.;Ethanol as a liquid fuel is a promising alternative to gasoline. However, corn-based ethanol consumes large amounts of corn, which is one of the main sources of human food. Cellulose is a very abundant potential feedstock for ethanol production, but is difficult to hydrolyze due to its crystal structure. Hydrolysis of cellulose is usually accomplished only by anaerobic bacteria. This process is slow and expensive. In the third project, brevibacillus sp. JXL, a novel aerobic, thermophilic, cellulose-degrading bacterium, was studied. Dynamic IEF was used to separate the cellulase from other proteins produced by JXL. The active cellulase was isolated within a five volt section (out of 2000 applied volts) using dynamic IEF, providing a resolution of ∼0.0175 pH and eliminating 99.75% of the inactive portion of the sample.