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.

Yes / Lichenicolous (lichen-dwelling) fungi have been extensively researched taxonomically over many years, and phylogenetically in recent years, but the biology of the relationship between the invading fungus and the lichen host has received limited attention, as has the effects on the chemistry of the host, being difficult to examine in situ. Raman spectroscopy is an established method for the characterization of chemicals in situ, and this technique is applied to a lichenicolous fungus here for the first time. Xanthoriicola physciae occurs in the apothecia of Xanthoria parietina, producing conidia at the hymenium surface. Raman spectroscopy of apothecial sections revealed that parietin and carotenoids were destroyed in infected apothecia. Those compounds protect healthy tissues of the lichen from extreme insolation and their removal may contribute to the deterioration of the apothecia. Scytonemin was also detected, but was most probably derived from associated cyanobacteria. This work shows that Raman spectroscopy has potential for investigating changes in the chemistry of a lichen by an invading lichenicolous fungus. / This work was completed while D.L.H. was in receipt of an award from the Ministerio de Economica y Competitividad of Spain (Proyectos CGL 2014-55542-P).

No / Scytonemin-imine, a novel derivative of scytonemin, has been isolated and identified very recently and proposed to serve as a photoprotective biomarker for certain bacteria growing under intense photon flux density. This study predicts theoretically the Raman spectrum of scytonemin-imine by density functional theory calculations and provides comparison of major bands to those of scytonemin, the parent compound for which both the experimentally characterized and theoretically predicted spectra exist in the literature. It is proposed to be an addendum to the collection of our previous work on scytonamin and its derivatives to facilitate recognition of the diagnostic Raman spectral signatures for scytonemin-imine.

No / Raman spectroscopy has been identified as an important first-pass analytical technique for deployment on planetary surfaces as part of a suite of instrumentation in projected remote space exploration missions to detect extant or extinct extraterrestrial life signatures. Aside from the demonstrable advantages of a non-destructive sampling procedure and an ability to record simultaneously the molecular signatures of biological, geobiological and geological components in admixture in the geological record, the interrogation and subsequent interpretation of spectroscopic data from these experiments will be critically dependent upon the recognition of key biomolecular markers indicative of life existing or having once existed in extreme habitats. A comparison made with the characteristic Raman spectral wavenumbers obtained from standards is not acceptable because of shifts that can occur in the presence of other biomolecules and their host mineral matrices. In this paper, we identify the major sources of difficulty experienced in the interpretation of spectroscopic data centring on a key family of biomarker molecules, namely scytonemin and its derivatives; the parent scytonemin has been characterized spectroscopically in cyanobacterial colonies inhabiting some of the most extreme terrestrial environments and, with the support of theoretical calculations, spectra have been predicted for the characterization of several of its derivatives which could occur in novel extraterrestrial environments. This work will form the foundation for the identification of novel biomarkers and for their Raman spectroscopic discrimination, an essential step in the interpretation of potentially complex and hitherto unknown biological radiation protectants based on the scytoneman and scytonin molecular skeletons which may exist in niche geological scenarios in the surface and subsurface of planets and their satellites in our Solar System.