Matrix assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOFMS) is used to study several inorganic coordination complexes and a variety of compounds from cyanobacteria. Also presented is a discussion of TOFMS instrumentation and the improvements in resolution and instrument automation that have lead to widespread and diverse applications of MALDI-TOFMS in all areas of science. The feasibility of using direct laser desorption/ionization (LDI) TOFMS to detect trace elements in a variety of glass samples using a lithium borate fusion technique for sample preparation is investigated. The result of the fusion technique is a homogeneous incorporation of the analytical sample into a glass. The fusion technique is investigated as a way to eliminate matrix effects in direct LDI-TOFMS analysis. However, the high concentration and low ionization potential of lithium suppress ionization of the elements of interest. The detection limits of elements in glass samples were not at the trace level. Therefore the technique is not as useful as well-established analytical methods like X-ray fluorescence and inductively coupled plasma mass spectrometry. Direct laser ablation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of four inorganic coordination complexes are discussed. The compounds studied include [Ir(dpp)2Cl2](PF6), {[(bpy)2Ru(dpp)]2RuCl2}(PF6)4, [(tpy)Ru(tpp)Ru(tpp)RhCl3](PF6)4 and {[(bpy)2Ru(dpp)]2IrCl2}(PF6)5 (dpp = 2,3-bis-(2'-pyridyl)-pyrazine, bpy = 2,2'-bipyridine, tpy = 2,2',6',2"-terpyradine, tpp = 2,3,5,6,-tetrakis-(2'-pyridyl)-pyrazine). Spectral intensities and fragmentation patterns are compared and evaluated for several instrument parameters, matrices, and matrix-to-analyte ratios. Direct ablation and MALDI mass spectra of the monometallic complex showed the same ion peaks and differed only in the relative peak intensities. Direct ablation of the trimetallic complexes produced only low-mass fragments containing one metal atom at most. MALDI spectra of the trimetallic complexes exhibited little fragmentation in the high-mass region (>1500 Da) and less fragmentation in the low-mass region compared to direct laser ablation. Proper matrix selection for MALDI analysis was vital, as was an appropriate matrix-to-analyte ratio. The results demonstrate the applicability of MALDI-TOF mass spectrometry for the structural characterization of labile inorganic coordination complexes. A correlation is made between the gas-phase redox chemistry in the MALDI plume and the solution phase electrochemistry for this series of complexes. MALDI-TOFMS was also used to study compounds isolated from cyanobacteria. A MALDI screening method has been developed to detect the presence of scytonemin, a UV-absorbing pigment. Detection of scytonemin is accomplished by a simple solvent extraction of cyanobacteria in the desiccated state with subsequent MALDI-TOFMS analysis. The method is rapid and semi-quantitative. Cyanobacteria is the only known organism to produce scytonemin, and it is only produced when the organism is subjected to UV stress. Laboratory-grown cultures were subjected to different amounts of UV radiation, and the screening method was used to detect the presence or absence of scytonemin. Cultures grown under ambient conditions (low UV) did not show the presence of scytonemin, while those grown under UV lamps did show the detectable scytonemin. Because scytonemin acts as a biomarker for UV stress, the MALDI screening method could find application in molecular ecology studies of cyanobacteria. Peptide mass fingerprinting is used to monitor the isolation of the water stress protein from N. commune. The protein is produced by recombinant growth in E. coli in order to assess the role of Wsp in the desiccation tolerance of N. commune. The results show that SDS-PAGE and Western blot analysis are not sufficient to detect the presence of Wsp after purification using ion-exchange chromatography. Three E. coli proteins were identified in the same molecular weight range as Wsp and one of them cross-reacts with the series of antibodies used for the Western blot. The presence of contaminating proteins that cross-react with the immuno assay make it difficult to determine which fractions contained Wsp. Peptide mass fingerprints were obtained for a series of fractions collected after ion-exchange chromatography to pinpoint the location of Wsp. Peptide mass fingerprinting was also used to monitor the stability of the clone and results show that the clone is modified over a six month period. / Ph. D.

Peptide mass fingerprinting (PMF) of protein digests is a widely-accepted method for protein identification in MS-based proteomic studies. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is the technique of choice in PMF experiments. The success of protein identification in a PMF experiment is directly related to the amount of amino acid sequence coverage. In an effort to increase the amount of sequence information obtained in a MALDI PMF experiment, performic acid oxidation is performed on tryptic digests of known proteins. Performic acid was chosen as the chemical oxidant due to the ease of use and to the selective oxidation of cysteine, methionine, and tryptophan residues. In experiments performed in our laboratory, performic acid oxidation either increased or did not affect protein sequence coverage in PMF experiments when oxidized tryptic digests were analyzed by MALDI. Negative mode MALDI data were acquired, as well as positive mode MALDI data, due to the enhanced ionization of cysteic acid-containing peptides in negative mode. Furthermore, the confidence in a protein match is increased by observation of mass shifts indicative of cysteine, methionine, and/or tryptophan in oxidized peptide ion signals when comparing MALDI spectra prior to performic acid oxidation and after oxidation due to the low abundance of these residues in the majority of all known and hypothetical proteins.

Peptide mass fingerprinting (PMF) of protein digests is a widely-accepted method for protein identification in MS-based proteomic studies. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) is the technique of choice in PMF experiments. The success of protein identification in a PMF experiment is directly related to the amount of amino acid sequence coverage. In an effort to increase the amount of sequence information obtained in a MALDI PMF experiment, performic acid oxidation is performed on tryptic digests of known proteins. Performic acid was chosen as the chemical oxidant due to the ease of use and to the selective oxidation of cysteine, methionine, and tryptophan residues. In experiments performed in our laboratory, performic acid oxidation either increased or did not affect protein sequence coverage in PMF experiments when oxidized tryptic digests were analyzed by MALDI. Negative mode MALDI data were acquired, as well as positive mode MALDI data, due to the enhanced ionization of cysteic acid-containing peptides in negative mode. Furthermore, the confidence in a protein match is increased by observation of mass shifts indicative of cysteine, methionine, and/or tryptophan in oxidized peptide ion signals when comparing MALDI spectra prior to performic acid oxidation and after oxidation due to the low abundance of these residues in the majority of all known and hypothetical proteins.

In this study, the soluble protein fraction of P. chrysosporium grown under standard conditions was analyzed by using 2D-PAGE approach and a 2-D reference map was constructed. 910 spots could be separated and detected on Coomassie-stained 2-D gels by the help of Delta2D image analysis software. 720 spots could be cut from the master gel and were subjected to MALDI-TOF MS analysis followed by MASCOT search. A total of 517 spots out of 720 were assigned to specific accession numbers from the P. chrysosporium genome database. Further analysis of the data revealed 314 different gene products (distinct ORFs). The theoretical pI and MW values were plotted against the experimental migration distances. Results indicated the existence of 124 protein spots whose horizontal migration differed significantly from the expected migration according to the calculated pI values and 52 spots with an apparent molecular weight that is significantly different from their theoretical molecular weight. While protein modification could be predicted by these analyses, the main support was the presence of multiple spots of the same gene product. As much as 118 ORFs yielded multiple spots on the master gel, corresponding to 37.5% of the all distinct ORFs identified in this work. The relative abundance of each of the 517 identified polypeptides was calculated in terms of spot intensity. The majority of the most abundant proteins were found to be housekeeping ones. When the relative distribution of the proteins into four main functional categories was taken into consideration, &ldquo / Metabolism&rdquo / appeared the most important category with a share of 50.6% among identified proteins. However, among the functional classes, &ldquo / Posttranslational modifications, protein turnover, chaperones&rdquo / which is listed under the main category &ldquo / Cellular Processing and Signalling&rdquo / was represented by the highest number (104) of the identified proteins. Only 6 of the proteins listed in this study were assigned to hypothetical proteins. Out of the 314 identified gene products shown in P. chrysosporium, 29 were predicted to have a signal peptide sequence according to the SignalP algorithm. By making a WoLF PSORT search, subcellular localization of the proteins was predicted. Accordingly, 147 of the proteins were predicted to be located in cytoplasm. The phosphorylated proteins of P. chrysosporium were detected by ProQ phosphoprotein staining of the 2-D gel. 380 out of 910 distinct protein spots (40%) were found to be phosphorylated in exponentially growing cells of P. chrysosporium. Of these spots, 96 could be matched to the identified proteins.

The goal of my diploma thesis was finding a suitable method for unifying score values from various protein identification search tools in MS/MS mass spectrometry into one single score value. Data coming from the output of mass spectrometer are processed in two independent search tools Mascot and X!Tandem. These were selected especially for their wide usage in proteomic labs. Both results are evaluated through newly designed function and unified by single valued score clearly identifying found proteins. Newly designed scoring value is called Matascore and function producing this score was implemented in MATLAB. Function and its results were successfully tested by real data available in public databases on the Internet.

No / Despite being widely utilized in the production of cultural objects, keratinous hard tissues, such as horn, baleen, and tortoiseshell, rarely survive in archaeological contexts unless factors combine to inhibit biodeterioration. Even when these materials do survive, working, use, and diagenetic changes combine to make identification difficult. This paper reviews the chemistry and deterioration of keratin and past approaches to the identification of keratinous archaeological remains. It describes the formation of horn, hoof, baleen, and tortoiseshell and demonstrates how identification can be achieved by combining visual observation under low-power magnification with an understanding of the structure and characteristic deterioration of these materials. It also demonstrates how peptide mass fingerprinting of the keratin can be used to identify keratinous tissues, often to species, even when recognizable structural information has not survived.