Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Differentiation of the Dimorphic Fungal Species Paracoccidioides brasiliensis and Paracoccidioides lutzii

Isolates of Paracoccidioides brasiliensis and Paracoccidioides lutzii, previously characterized by molecular techniques, were identified for the first time by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). All isolates were correctly identified, with log score values of >2.0. Thus, MALDI-TOF MS is a new tool for differentiating species of the genus Paracoccidioides.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Characterization of Primary Amine End-Functionalized Polystyrene and Poly(methyl methacrylate) Synthesized by Living Anionic Polymerization Techniques

The reaction of living anionic polymers with 2,2,5,5-tetramethyl-1-(3-bromopropyl)-1-aza-2,5- disilacyclopentane (1) was investigated using coupled thin layer chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of byproducts as well as the major product were determined. The anionic initiator having a protected primary amine functional group, 2,2,5,5-tetramethyl- 1-(3-lithiopropyl)-1-aza-2,5-disilacyclopentane (2), was synthesized using all-glass high-vacuum techniques, which allows the long-term stability of this initiator to be maintained. The use of 2 in the preparation of well-defined aliphatic primary amine R-end-functionalized polystyrene and poly(methyl methacrylate) was investigated. Primary amino R-end-functionalized poly(methyl methacrylate) can be obtained near-quantitatively by reacting 2 with 1,1-diphenylethylene in tetrahydrofuran at room temperature prior to polymerizing methyl methacrylate at -78 °C. When 2 is used to initiate styrene at room temperature in benzene, an additive such as N,N,N',N'- tetramethylethylenediamine is necessary to activate the polymerization. However, although the resulting polymers have narrow molecular weight distributions and well-controlled molecular weights, our mass spectra data suggest that the yield of primary amine α-end-functionalized polystyrene from these syntheses is very low. The majority of the products are methyl α-end-functionalized polystyrene.

Fenofibrate metabolism in the cynomolgus monkey using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry-based metabolomics

Fenofibrate, widely used for the treatment of dyslipidemia, activates the nuclear receptor, peroxisome proliferator-activated receptor alpha. However, liver toxicity, including liver cancer, occurs in rodents treated with fibrate drugs. Marked species differences occur in response to fibrate drugs, especially between rodents and humans, the latter of which are resistant to fibrate-induced cancer. Fenofibrate metabolism, which also shows species differences, has not been fully determined in humans and surrogate primates. In the present study, the metabolism of fenofibrate was investigated in cynomolgus monkeys by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS)-based metabolomics. Urine samples were collected before and after oral doses of fenofibrate. The samples were analyzed in both positive-ion and negative-ion modes by UPLC-QTOFMS, and after data deconvolution, the resulting data matrices were subjected to multivariate data analysis. Pattern recognition was performed on the retention time, mass/charge ratio, and other metabolite-related variables. Synthesized or purchased authentic compounds were used for metabolite identification and structure elucidation by liquid chromatographytandem mass spectrometry. Several metabolites were identified, including fenofibric acid, reduced fenofibric acid, fenofibric acid ester glucuronide, reduced fenofibric acid ester glucuronide, and compound X. Another two metabolites (compound B and compound AR), not previously reported in other species, were characterized in cynomolgus monkeys. More importantly, previously unknown metabolites, fenofibric acid taurine conjugate and reduced fenofibric acid taurine conjugate were identified, revealing a previously unrecognized conjugation pathway for fenofibrate.

Performance evaluation of a miniature laser ablation time-of-flight mass spectrometer designed for in situ investigations in planetary space research

Key performance features of a miniature laser ablation time-of-flight mass spectrometer designed for in situ investigations of the chemical composition of planetary surfaces are presented. This mass spectrometer is well suited for elemental and isotopic analysis of raw solid materials with high sensitivity and high spatial resolution. In this study, ultraviolet laser radiation with irradiances suitable for ablation (< 1 GW/cm2) is used to achieve stable ion formation and low sample consumption. In comparison to our previous laser ablation studies at infrared wavelengths, several improvements to the experimental setup have been made, which allow accurate control over the experimental conditions and good reproducibility of measurements. Current performance evaluations indicate significant improvements to several instrumental figures of merit. Calibration of the mass scale is performed within a mass accuracy (Δm/m) in the range of 100 ppm, and a typical mass resolution (m/Δm) ~600 is achieved at the lead mass peaks. At lower laser irradiances, the mass resolution is better, about (m/Δm) ~900 for lead, and limited by the laser pulse duration of 3 ns. The effective dynamic range of the instrument was enhanced from about 6 decades determined in previous study up to more than 8 decades at present. Current studies show high sensitivity in detection of both metallic and non-metallic elements. Their abundance down to tens of ppb can be measured together with their isotopic patterns. Due to strict control of the experimental parameters, e.g. laser characteristics, ion-optical parameters and sample position, by computer control, measurements can be performed with high reproducibility. Copyright © 2012 John Wiley & Sons, Ltd.

Protein quaternary structure determination using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemical crosslinking

A means of analyzing protein quaternary structure using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI MS) and chemical crosslinking was evaluated. Proteins of known oligomeric structure, as well as monomeric proteins, were analyzed to evaluate the method. The quaternary structure of proteins of unknown or uncertain structure was investigated using this technique. The stoichiometry of recombinant E. coli carbamoyl phosphate synthetase and recombinant human farnesyl protein transferase were determined to be heterodimers using glutaraldehyde crosslinking, agreeing with the stoichiometry found for the wild type proteins. The stoichiometry of the gamma subunit of E. coli DNA polymerase III holoenzyme was determined in solution without the presence of other subunits to be a homotetramer using glutaraldehyde crosslinking and MALDI MS analysis. Chi and psi subunits of E. coli DNA polymerase III subunits appeared to form a heterodimer when crosslinked with heterobifunctional photoreactive crosslinkers.^ Comparison of relative % peak areas obtained from MALDI MS analysis of crosslinked proteins and densitometric scanning of silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels showed excellent qualitative agreement for the two techniques, but the quantitative analyses differed, sometimes significantly. This difference in quantitation could be due to SDS-PAGE conditions (differential staining, loss of sample) or to MALDI MS conditions (differences in ionization and/or detection). Investigation of pre-purified crosslinked monomers and dimers recombined in a specific ratio revealed the presence of mass discrimination in the MALDI MS process. The calculation of mass discrimination for two different MALDI time-of-flight instruments showed the loss of a factor of approximately 2.6 in relative peak area as the m/z value doubles over the m/z range from 30,000 to 145,000 daltons.^ Indirect symmetry was determined for tetramers using glutaraldehyde crosslinking with MALDI MS analysis. Mathematical modelling and simple graphing allowed the determination of the symmetry for several tetramers known to possess isologous D2 symmetry. These methods also distinguished tetramers that did not fit D2 symmetry such as apo-avidin. The gamma tetramer of E. coli DNA polymerase III appears to have isologous D2 symmetry. ^

A new Time-Of-Flight Mass Spectrometer (TOF-MS) for noble gas analysis

Noble gas analysis in early solar system materials, which can provide valuable information about early solar system processes and timescales, are very challenging because of extremely low noble gas concentrations (ppt). We therefore developed a new compact sized (33 cm length, 7.2cm diameter, 1.3 L internal volume) Time-of-Flight (TOF) noble gas mass spectrometer for high sensitivity. We call it as Edel Gas Time-of-flight (EGT) mass spectrometer. The instrument uses electron impact ionization coupled to an ion trap, which allows us to ionize and measure all noble gas isotopes. Using a reflectron set-up improves the mass resolution. In addition, the reflectron set-up also enables some extra focusing. The detection is via MCPs and the signals are processed either via ADC or TDC systems. The objective of this work is to understand the newly developed Time-Of-Flight (TOF) mass spectrometer for noble gas analysis in presolar grains of the meteorites. Chapter 1 briefly introduces the basic idea and importance of the instrument. The physics relevant to time-of-flight mass spectrometry technique is discussed in the Chapter 2 and Chapter 3 will present the oxidation technique of nanodiamonds of the presolar grains by using copper oxide. Chapter 4 will present the details about EGT data analysis software. Chapter 5 and Chapter 6 will explain the details about EGT design and operation. Finally, the performance results will be presented and discussed in the Chapter 7, and whole work is summarized in Chapter 8 and also outlook of the future work is given.

Sensitivity and Fragmentation Calibration of the ROSINA Reflectron-type Time-Of-Flight Mass Spectrometer

The goal of this work was to increase the performance and to calibrate one of the ROSINA sensors, the Reflectron-type Time-Of-Flight mass spectrometer, currently flying aboard the ESA Rosetta spacecraft. Different optimization techniques were applied to both the lab and space models, and a static calibration was performed using different gas species expected to be detected in the vicinity of comet 67P/Churyumov-Gerasimenko. The database thus created was successfully applied to space data, giving consistent results with the other ROSINA sensors.

DNA sequencing by delayed extraction-matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a delayed ion extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.

Peptide quantification by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry: Investigations of the cyclotide kalata B1 in biological fluids

A rapid method has been developed for the quantification of the prototypic cyclotide kalata B I in water and plasma utilizing matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. The unusual structure of the cyclotides means that they do not ionise as readily as linear peptides and as a result of their low ionisation efficiency, traditional LC/MS analyses were not able to reach the levels of detection required for the quantification of cyclotides in plasma for pharmacokinetic studies. MALDI-TOF-MS analysis showed linearity (R-2 > 0.99) in the concentration range 0.05-10 mu g/mL with a limit of detection of 0.05 mu g/mL (9 fmol) in plasma. This paper highlights the applicability of MALDI-TOF mass spectrometry for the rapid and sensitive quantification of peptides in biological samples without the need for extensive extraction procedures. (c) 2005 Elsevier B.V. All rights reserved.