Detection and quantification of protein oxidation in sarcopenic models:a mass spectrometry study

Oxidised biomolecules in aged tissue could potentially be used as biomarkers for age-related diseases; however, it is still unclear whether they causatively contribute to ageing or are consequences of the ageing process. To assess the potential of using protein oxidation as markers of ageing, mass spectrometry (MS) was employed for the identification and quantification of oxidative modifications in obese (ob/ob) mice. Lean muscle mass and strength is reduced in obesity, representing a sarcopenic model in which the levels of oxidation can be evaluated for different muscular systems including calcium homeostasis, metabolism and contractility. Several oxidised residues were identified by tandem MS (MS/MS) in both muscle homogenate and isolated sarcoplasmic reticulum (SR), an organelle that regulates intracellular calcium levels in muscle. These modifications include oxidation of methionine, cysteine, tyrosine, and tryptophan in several proteins such as sarcoplasmic reticulum calcium ATPase (SERCA), glycogen phosphorylase, and myosin. Once modifications had been identified, multiple reaction monitoring MS (MRM) was used to quantify the percentage modification of oxidised residues within the samples. Preliminary data suggests proteins in ob/ob mice are more oxidised than the controls. For example SERCA, which constitutes 60-70% of the SR, had approximately a 2-fold increase in cysteine trioxidation of Cys561 in the obese model when compared to the control. Other obese muscle proteins have also shown a similar increase in oxidation for various residues. Further analysis with complex protein mixtures will determine the potential diagnostic use of MRM experiments for analysing protein oxidation in small biological samples such as muscle needle biopsies.

The Development of High Performance Liquid Chromatography Systems for the Analysis of Improvised Explosives

Existing instrumental techniques must be adaptable to the analysis of novel explosives if science is to keep up with the practices of terrorists and criminals. The focus of this work has been the development of analytical techniques for the analysis of two types of novel explosives: ascorbic acid-based propellants, and improvised mixtures of concentrated hydrogen peroxide/fuel. In recent years, the use of these explosives in improvised explosive devices (IEDs) has increased. It is therefore important to develop methods which permit the identification of the nature of the original explosive from post-blast residues. Ascorbic acid-based propellants are low explosives which employ an ascorbic acid fuel source with a nitrate/perchlorate oxidizer. A method which utilized ion chromatography with indirect photometric detection was optimized for the analysis of intact propellants. Post-burn and post-blast residues if these propellants were analyzed. It was determined that the ascorbic acid fuel and nitrate oxidizer could be detected in intact propellants, as well as in the post-burn and post-blast residues. Degradation products of the nitrate and perchlorate oxidizers were also detected. With a quadrupole time-of-flight mass spectrometer (QToFMS), exact mass measurements are possible. When an HPLC instrument is coupled to a QToFMS, the combination of retention time with accurate mass measurements, mass spectral fragmentation information, and isotopic abundance patterns allows for the unequivocal identification of a target analyte. An optimized HPLC-ESI-QToFMS method was applied to the analysis of ascorbic acid-based propellants. Exact mass measurements were collected for the fuel and oxidizer anions, and their degradation products. Ascorbic acid was detected in the intact samples and half of the propellants subjected to open burning; the intact fuel molecule was not detected in any of the post-blast residue. Two methods were optimized for the analysis of trace levels of hydrogen peroxide: HPLC with fluorescence detection (HPLC-FD), and HPLC with electrochemical detection (HPLC-ED). Both techniques were extremely selective for hydrogen peroxide. Both methods were applied to the analysis of post-blast debris from improvised mixtures of concentrated hydrogen peroxide/fuel; hydrogen peroxide was detected on variety of substrates. Hydrogen peroxide was detected in the post-blast residues of the improvised explosives TATP and HMTD.

Biodegradation of acrylamide by acrylamidase from Stenotrophomonas acidaminiphila MSU12 and analysis of degradation products by MALDI-TOF and HPLC

The present study examines an improved detoxification and rapid biological degradation of toxic pollutant acrylamide using a bacterium. The acrylamide degrading bacterium was isolated from the soil followed by its screening to know the acrylamide degrading capability. The minimal medium containing acrylamide (30 mM) served as a sole source of carbon and nitrogen for their active growth. The optimization of three different factors was analyzed by using Response Surface Methodology (RSM). The bacteria actively degraded the acrylamide at a temperature of 32 degrees C, with a maximum growth at 30 mM substrate (acrylamide) concentration at a pH of 7.2. The acrylamidase activity and degradation of acrylamide was determined by High Performance Liquid Chromatography (HPLC) and Matrix Assisted Laser Desorption and Ionization Time of Flight mass spectrometer (MALDI-TOF). Based on 168 rRNA analysis the selected strain was identified as Gram negative bacilli Stenotrophomonas acidaminiphila MSU12. The acrylamidase was isolated from bacterial extract and was purified by HPLC, whose mass spectrum showed a molecular mass of 38 kDa. (C) 2014 Elsevier Ltd. All rights reserved.

Kinetic energies of C-n(+) (n <= 58) fragment ions produced by nanosecond laser impact on C-60

Laser-induced fragmentation of C-60 has been studied using a time-of-flight mass spectrometric technique. The average kinetic energies of fragment ions C-n(+) (n <= 58) have been extracted from the measured full width at half maximum (FWHM) of ion beam profiles. The primary formation mechanism of small fragment ion C-n(+) (n < 30) is assumed to be a two-step fragmentation process: C60 sequential decay to unstable C-30(+) ion and the binary fission of C-30(+). Considering a second photo absorption process in the later part of laser pulse duration, good agreement is achieved between experiment and theoretical description of photoion formation. (C) 2009 Elsevier B. V. All rights reserved.

Dynamics of excited m-dichlorobenzene

Dynamics of excited m-dichlorobenzene is investigated in real time by femtosecond pump-probe method, combined with time-of-flight mass spectrometric detection in a supersonic molecular beam. The yields of the parent ion and daughter ion C6H4CI+ are examined as a function of the delay between the 270 and 810 nm femtosecond laser pulses, respectively. The lifetime of the first singlet excited state S-1 of m-dichlorobenzene is measured. The origin of this daughter ion C6H4CI+ is discussed. The ladder mechanism is proposed to form the fragment ion. In addition, our experimental results exhibit a rapid damped sinusoidal oscillation over intermediate time delays, which is due to quantum beat effects.

Multiphoton ionization of pyrrole-water mixed clusters

Multiphoton ionization of the hydrogen,bonded pyrrole-water clusters (C4H5N)(n)(H2O)(m) is studied with a reflectron-time of flight mass spectrometer at 355 mn. With increasing partial concentration of pyrrole in a gas mixture source, a series of poly-pyrrole-water binary-mixed cluster ions can be observed, including unprotonated cluster ions [(C4H5N)(x)(H2O)(y)](+), protonated cluster ions [(C4H5N)(x)(H2O)(y)](+) and dehydrogenated cluster ions [(C4H4N)(C4H5N)(x)(H2O)(y)](+). Ab initio calculations of their structures, bond strengths, charge distributions and reaction energies are carried out. Stable structures of these clusters are obtained from the calculations. A probable formation mechanism of the cluster ions [(C4H5N)(x)(H2O)(y)](+), [(C4H5N)(x)(H2O)(y)]H+ and [(C4H4N)(C4H5N)(x) (H2O)(y)](+) is supposed to be the ionization of clusters followed by dissociation.

Decay dynamics of excited CS2 and NH3 investigated by femtosecond laser

By using the home-made femtosecond laser system and the time-of-flight mass spectrometer, the decay dynamics of excited carbon disulfide (CS2) and ammonia (NH3) are investigated in real time by pump-probe multiphoton ionization detection. The estimated lifetime constant of the NH3 (A) over tilde (1)A(2)' state (51+/-4 fs) agreed quite well with the literature report. For the first time, the decay lifetime constants of the NH3 (E) over tilde'(1)A(1)' state (937+/-93 fs), the CS2 (a) over tilde (3)A(2) state (153+/-10 fs), and the CS2 Rydberg state [(3)/(2)]6ssigma(g) ((3)Pi(g)) (948+/-23 fs) are obtained.

New high-lying odd-parity excited levels of atomic uranium

We report the measurement of 112 new high-lying odd-parity excited levels of U I in the energy region 35 678-36 696 cm(-1). These levels were obtained with a setup composed of a Nd:YAG-laser-pumped pulsed dye laser system, an atomic beam device, a time-of-flight mass spectrometer, and a boxcar integrator. (C) 2000 Optical Society of America [S0740-3224(99)02309-7] OCIS code: 300.0300.

A simple method for the selective enrichment of endohedral metallofullerenes

The concentration of a polar solvent DMF extract was found to be very effective for the selective enrichment of endohedral metallofullerenes against empty fullerenes. As the solvent evaporated, endohedral metallofullerenes were effectively enriched in the solution, while most of empty fullerenes (especially C-60 and C-70) were precipitated because of their scant solubility in DMF. Matrix-assisted laser-desorption-ionization time-of-fligh mass spectrometry analysis indicated that the purity of endohedral metallofullerenes increased dramatically after concentration of the DMF extract solution. Upon transferring the extract into toluene, a solution containing significantly enriched endohedral metallofullerenes was obtained. The different solubilities of endohedral metallofullerenes versus empty fullerenes are considered to account for this selective enrichment of endohedral metallofullerenes.

Formation and dissociation of protonated pyridine-methanol cluster ions at 355 and 266 nm

The multi-photon ionization process of the hydrogen-bond cluster of pyridine-methanol has been investigated using a conventional and reflectron time-of-flight mass spectrometer (RTOF-MS) at 355 and 266 nm laser wavelengths, respectively. The sequences of the protonated cluster ions (CH3OH)(n)H+ and (C5H5Nn)(CH3OH)(m)H+ (n = 1,2) were observed at both laser wavelengths, while the sequence of the cluster ions (CH3)OHn (H2O)H+ was observed only at 355 nm laser wavelength. The difference between the relative signal intensities of the protonated methanol cluster ions at different laser wavelengths is attributed to different photoionization mechanisms. Some nascent cluster ions in metastable states dissociated during free flight to the detector. The dissociation kinetics is also discussed. (C) 2000 Elsevier Science B.V.

The ion-molecule reaction after multiphoton ionization in the binary cluster of ammonia and methanol

The binary cluster (CH3OH)(n)(NH3)(m) was studied by using a multiphoton ionization time-of-flight mass spectrometer (MPI-TOFMS). The measured two series of protonated cluster ions: (CH3OH)(n)H+ and (CH3OH)(n)NH4+ (1 less than or equal to n less than or equal to 14) were attributed to the ion-molecule reaction in the binary cluster ions. The mixed cluster of CH3OD with ammonia was also studied. The results implied that the proton transfer probability from the OD group was larger than that from CH3 group. The ab initio calculation of the binary cluster was carried out at the HF/STO-3G and MP2/6-31G** levels of theory, and indicated that the latter process of the proton transfer must overcome a barrier of similar to 29 kcal/mol. (C) 1999 Elsevier Science B.V. All rights reserved.

Experimental observation of dissociative electron attachment to S2O and S2O2 with a new spectrometer for unstable molecules

A new spectrometer, electron radical interaction chamber, has been developed to study dissociative electron attachment to unstable molecules such as free radicals. It includes a trochoidal electron monochromator and a time-of-flight mass spectrometer. Radicals are generated with a microwave discharge at 2.45 GHz. Preliminary data are presented for radicals formed when a mixture of helium and sulphur dioxide was passed through the microwave discharge. Several new resonances are observed with the discharge on. Resonances at 0 eV (S-), 0.8, 1.2, 3.0 eV (SO-) and 3.7 eV (SO- and S2O-) are assigned to the radical S2O2 and a resonance at 1.6 eV (S-) is assigned to S2O. No new resonances have been assigned to SO, which was also generated in the microwave discharge.

Isolation of AF2 (KHEYLRFamide) from Caenorhabditis elegans: Evidence for the presence of more than one FMRFamide related peptide-encoding gene

Numerous FMRF amide-related peptides (FaRPs) have been isolated and sequenced from extracts of free-living and parasitic nematodes. The most abundant FaRP identified in ethanolic/methanolic extracts of the parasitic forms, Ascaris suum and Haemonchus contortus and from the free-living nematode, Panagrellus redivivus, was KHEYLRF amide (AF2). Analysis of the nucleotide sequences of cloned FaRP-precursor genes from C. elegans and, more recently, Caenorhabditis vulgaris identified a series of related FaRPs which did not include AF2. An acid-ethanol extract of Caenorhabditis elegans was screened radioimmunometrically for the presence of FaRPs using a C-terminally directed FaRP antiserum. Approximately 300 pmols of the most abundant immunoreactive peptide was purified to homogeneity and 30 pmols was subjected to Edman degradation analysis and gas-phase sequencing. The unequivocal primary structure of the heptapeptide, Lys-His-Glu-Tyr-Leu-Arg-Phe-NH2 (AF2) was determined following a single gas-phase sequencing run. The molecular mass of the peptide was determined using a time-of-flight mass spectrometer and was found to be 920 (MH(+))(-), which was consistent with the theoretical mass of C-terminally amidated AF2. These results indicate that C. elegans possesses more than one FaRP gene. (C) 1995 Academic Press, Inc.

Detection Limits of Organic Compounds Achievable with Intense, Short-Pulse Lasers

Many organic molecules have strong absorption bands which can be accessed by ultraviolet short pulse lasers to produce efficient ionization. This resonant multiphoton ionization scheme has already been exploited as an ionization source in time-of-flight mass spectrometers used for environmental trace analysis. In the present work we quantify the ultimate potential of this technique by measuring absolute ion yields produced from the interaction of 267 nm femtosecond laser pulses with the organic molecules indole and toluene, and gases Xe, N2 and O2. Using multiphoton ionization cross sections extracted from these results, we show that the laser pulse parameters required for real-time detection of aromatic molecules at concentrations of one part per trillion in air and a limit of detection of a few attomoles are achievable with presently available commercial laser systems. The potential applications for the analysis of human breath, blood and tissue samples are discussed.

TAP Reactor Development and Application in the Kinetic Characterization of Catalysts for Heterogeneously Catalyzed Gas Phase Reactions

This manuscript describes the application and further development of the TAP technique in kinetic characterization of heterogeneous catalysis. The major application of TAP systems is to study mechanisms, kinetics and transport phenomena in heterogeneous catalysis, all of which is made possible by the sub-millisecond time resolution. Furthermore, the kinetic information obtained can be used to gain an insight into the mechanism occurring over the catalyst system. This is advantageous as heterogeneous catalysts with an improved efficiency can be developed as a result. TAP kinetic studies are carried out at low pressure (~1x10-7 mbar) and TAP pulses are sufficiently small (1013-1015 molecules) so as to maintain this low pressure. The use of a small number of molecules in comparison to the total number of active sites means the state of the catalyst remains relatively unchanged. The use of the low intensity pulses also makes the pressure gradient negligible and so allows the TAP reactor system to operate in the Knudsen Diffusion regime, where gas-gas reactions are eliminated. Hence only gas-catalyst reactions are investigated and, by the use of moment analysis of observed exit flow, rate constants of elementary steps of the reaction can be obtained.

In this manuscript, two attempts to further the TAP technique are reported. Firstly, the work undertaken at QUB to attempt to control the number of molecules of condensable reagents that can be pulsed during a TAP pulse experiment is disclosed. Secondly, a collaborative project with SAI Ltd Manchester is discussed in a separate chapter, where technical details and validation of a customised time of flight mass spectrometer (ToF MS) for the QUB TAP-1 system are reported. A collaborative project with Cardiff Catalysis Institute focusing on the study of CO oxidation over hopcalite catalysts is also reported. The analysis of the experimental results has provided an insight into the possible mechanism of the oxidation of CO over these catalysts. A correction function has also been derived which accounts for the adsorption of reactant molecules over inert materials that are used for the reactor packing in TAP experiments. This function was then applied to the selective reduction of O2 in a H2 rich ethene feed, so that more accurate TAP moment based analysis could be conducted.