Mark Domin

The Characterization of Micro-organisms by Matrix- assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

A variety of gram-positive and gram-negative intact bacterial cells have been analysed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and shown to provide fingerprint mass spectra with discrete peaks being observed over the mass range from 3 to 40 kDa. The spectra show both more peaks and peaks at a higher mass/charge ratio than have hitherto been reported for these micro-organisms and would appear to provide a profile of cellular proteinaceous material. The spectra are shown to be reproducible over variable time periods of up to three months and factors affecting reproducibility are discussed. The procedure, which requires minimal sample preparation, yields results in 30-40 minutes and allows visual identification of species- and strain-specific biomarkers for the characterization of the organisms. The importance of accurately defining sample preparation methodologies is central to the ability of the technique to generate reliable and reproducible data.

Molecular mass determinations in coal-derived liquids by MALDI mass spectrometry and size-exclusion chromatography

Abstract This study examined the effect of changes in instrument-related parameters on mass spectra obtained by MALDI-m.s.: ion-extraction voltage, laser power and sample loading. Results were compared with those of size-exclusion chromatography (s.e.c.) using 1-methyl-2-pyrrolidinone as mobile phase. A coal tar pitch and its pyridine-soluble and pyridine-insoluble fractions were used as samples. MALDI-mass spectra were obtained with no added matrix, since the sample absorbed strongly at the laser wavelength, 337 nm. Higher extraction voltages (up to 30 kV) led to the observation of higher masses in the whole pitch and its pyridine-insoluble fraction, where the presence of higher-molecular-mass material was indicated by s.e.c. Increasing the ion extraction voltage served only to accelerate species already ionized, without otherwise disturbing the sample: the use of higher ion extraction voltages must be therefore be considered as providing a more complete inventory of already ionized species. Increasing the laser power level and sample loading on the target led to similar changes in the spectra, although there were indications that for both these parameters, the results might no longer improve (and might eventually deteriorate) beyond optimum values, which appear to be sample-dependent. The low-power spectra revealed more structural information at low masses, where series of homologous peaks could be observed, compared with the spectra at high power.

The effect of solvent and matrix combinations on the analysis of bacteria by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

The ability to rapidly identify the taxonomic class of the wide variety of microorganisms involved in human and animal disease is becoming increasingly important, especially with the increasing development of resistance to the antibiotics which form the main defence against them. A number of groups have recognised the utility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry MALDI-TOF in the analysis of these microorganisms. However, no consistent methodology has been developed which is in general use. In particular the use of different solvent extraction systems and mass spectrometric matrices can have significant effects on the quality of the data obtained. We have now studied a number of the commonly used matrices and a range of solvent systems of widely varying polarity in an attempt to devise an optimum analytical strategy for the rapid characterisation of these organisms by MALDI-TOFMS. The E. coli ATCC 9637 organisms were initially washed to remove growth medium contaminants, followed by extraction with one of a range of solvents prior to admixing with a number of different single matrices or binary and ternary combinations of these matrices. The results obtained indicate that a binary combination of 2-(4-hydroxyphenylazo)benzoic acid and 2-mercaptobenzothiazole (1:1) as matrix provides the best data after the proteinaceous material from the organism cell surface was extracted with 17% formic acid, 33% isopropyl alcohol and 50% water, (solvent 2 in this work).

Matrix-assisted Laser Desorption/Ionization Mass Spectrometry of Pitch Fractions Separated by Planar Chromatography

Molecular mass distributions of coal-tar pitch fractions separated by planar chromatography (P-C) have been compared by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, carried out in the absence of added matrix. Solvent pairs used in the P-C separation were pyridine-acetonitrile, pyridine-N,ZVdimethylformamide and tetrahydrofuran-toluene. Molecular complexity and average molecular masses were found to increase with decreasing mobilities of the fractions. UV-fluorescence spectroscopy showed shifts of peak intensities to longer wavelengths and decreasing quantum yields, suggesting the presence of progressively greater concentrations of large polynuclear aromatic systems with decreasing mobility of samples in P-C. Fractions immobile in pyridine gave MALDI mass spectra of low intensity, indicating that larger molecules are less easily desorbed or ionized. The observed similarity of upper-mass limits of planar-chromatographic fractions of different mobility is thought to suggest the presence of relatively large-molecular mass (MM) materials of variable polarity in coal-tar pitch. The results were consistent with earlier investigations showing the presence of high molecular mass materials in coal-derived liquids. MM-distributions determined by size exclusion chromatography (SEC) were also observed to increase with decreusing mobility of the fractions on P-C plates. This qualitative agreement with results from MALDI-MS indicates that reported shifts of polar molecules to shorter elution times in SEC (i.e. to apparently larger-MMs) were not of a sufficient magnitude to distort the relative ordering of MM-distributions of PC-fractions observed by SEC. A number of problems relating to the refinement of MALDI-MS determinations on complex mixtures are discussed.

Absolute calibration of size exclusion chromatography for coal derivatives through MALDI-m.s.

This paper reports the fractionation and subsequent examination of two, very different coal derived materials, a coal tar pitch and a commercial liquefaction extract. These were separated on the basis of their retention in a typical preparative size exclusion chromatography apparatus using THF as the eluent. Matrix assisted laser desorption mass spectrometry, using the small molecules of the sample as matrix, has been used to examine the various fractions from preparative s.e.c. and the peak mass intensities have been used to define an absolute calibration of the s.e.c. separation. Limitations of the methods are discussed. The u.v.-fluorescence spectra of the fractions were recorded and trends in the spectra are described. The fractions were also analyzed by size exclusion chromatography employing N-methyl-2-pyrrolidinone as the chromatographic eluent. The main advantages of this system over traditional solvents is the avoidance of surface adsorption effects observed with THF and the more complete solution of coal-derived material.

Choice of Extraction Voltage and Matrix in the Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry of Coal Tar Pitch - Pyridine Insolubles †

Variations in molecular mass distributions observed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry as a function of changes in the ion accelerating voltage and composition of the matrix have been investigated, using the pyridine-insoluble fraction of a coal tar pitch. With increasing ion extraction voltages (10, 20 and 30 kV) and in the absence of added matrix, spectra clearly showed increases in intensity, particularly at higher masses. The use of higher ion extraction voltages may be considered as providing a more complete inventory of ionized species, apparently significantly enhancing the kinetic energy imparted to larger molecular weight materials. Magnitudes of molecular masses at maximum ion intensity observed using a set of different matrices may be ordered as follows: m/z 1500 [dimethoxy-4-hydroxycinnamic (sinapinic) acid], m/z 1400 (2,5-dihydroxybenzoic acid), m/z 900 [2-(4-hydroxyphenylazo)-benzoic acid], m/z 900 (AE-cyano-3-hydroxycinnamic acid), m/z 300 (9-anthracenecarboxylic acid) and m/z 300 (no matrix and a sample loading comparable with the with-matrix experiments). Maximum ion intensities, in the absence of matrix, were found at values which varied from about 300 m/z up to about m/z 1500 as sample loading varied from very low (i.e. suitable for with-matrix operation) up to a visibly black spot. In the absence of a quantitatively based criterion for distinguishing between signal and instrument noise at high mass, accurate high mass limits were not available. However, in each case, ion signals could clearly be observed at values greater than m/z 100 000. The present MALDI spectra do not necessarily show the highest molecular mass materials present in the sample. In view of the observed changes in the spectra with ion extraction voltage and matrix composition, it seems conceivable that higher voltages and/or specific matrices, other than those used in the study, may lead to the detection of larger molecules.

Effect of Polydispersity on the Characterization of Coal-derived Liquids by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry: Inferences from Results for Mixtures of Polystyrene Molecular Mass Standards

Seven polystyrene molecular mass standards have been run in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) singly, in pairs, in groups of three and as a mixture of the seven distinct samples. All standards had polydispersities of near unity. When run singly or in pairs, MALDI-spectra of all standard polymer samples could be observed clearly and showed approximately equal areas. Mixtures of multiple standards have been run at variable ion extraction voltages: when the voltage was reduced below 25 kV, low molar mass samples tended to predominate in the spectra, at the expense of the higher molar mass standards. Peaks due to the high mass standards were still observable above the noise level, but (for equimolar mixtures) areas under the peaks were no longer comparable. With the full set of seven samples, discrimination against intensities of the higher mass standards could be clearly observed, even at the highest ion extraction voltage available (30 kV). With increasing ion extraction voltage the MALDI-spectra of a coal tar pitch and its tetrahydrofuran-insoluble fraction showed increasing intensities of higher mass ranges. Due to the wide polydispersity of coal derived samples, MALDI-spectra would be expected to severely underestimate the proportion of high mass materials present. Further work on mixtures of different types of polymer standards would clearly be useful; for coal derived liquids, more extensive fractionation than that used here would seem advisable in order to reduce sample polydispersity.

Fractionation of a wood tar pitch by planar chromatography for the characterisation of large molecular mass materials

Abstract A commercial tar pitch derived from pine wood – Massen Pine ( Pinus Massonia ) and sold as Stockholm tar has been fractionated by planar chromatography with examination of the fractions by size exclusion chromatography in NMP eluent, by UV-fluorescence and by matrix assisted laser desorption mass spectrometry. The relatively small molecules, mobile in planar chromatography, are shown to be non-polar. Large molecules were found in each fraction, corresponding in SEC elution times up to polystyrenes of molecular mass of at least 1.8 million. Size exclusion chromatography profiles by UV light absorbance showed differences in relative absorbance of different wavelengths for large and small molecules, implying differences in structures. MALDI mass spectra indicated molecules of mass of several thousand mass units with the upper limit of mass not defined. Planar chromatography provides a fast, cheap method of isolating large molecular mass fractions of this biomass tar.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of kerogen extracts: effect of interactions between sample structure and matrix

This paper describes experiments carried out within the framework of examining capabilities of MALDI-TOF-MS in the characterisation of fossil fuels and related materials. The work has focused on the effect of interactions between sample structure and matrix composition on the quality of MALDI spectra. Solvent extracts from six kerogens have been examined by MALDI-MS in the presence of three matrices of different composition and in the absence of matrix (laser ablation). The kerogen extracts have also been characterised by size exclusion chromatography (SEC) and UV-fluorescence spectroscopy (UV-F). In the presence of the three matrices (sinapinic acid, 2,5-dihydroxybenzoic acid and AE-cyano-3-hydroxycinnamic acid), spectra of extracts from the three geologically ‘younger’ kerogens showed larger molecular mass distributions compared to spectra of extracts from the corresponding ‘older’ kerogens. This result directly contradicted trends observed in the absence of matrix, where extracts from ‘older’ kerogens showed larger molecular mass distributions. Results from SEC and UV-F agreed with trends observed in MALDI-spectra acquired in the presence of matrix, particularly sinapinic acid. By contrast, we recently reported that high mass signal in laser ablation mass spectra of the pyridine-insoluble fraction of a coal tar pitch, obtained in the absence of matrix, closely matched signals obtained in the presence of sinapinic acid. Compared to the highly condensed aromatic structure of the coal tar pitch, the kerogen extracts (of considerably higher oxygen content) appear less able to readily absorb incident laser radiation (337 nm) in the absence of externally added matrix. The argument is consistent with apparently more efficient absorption of incident laser radiation by the geologically ‘older’ samples, when no matrix was used. Structural characteristics suggest that the geologically ‘older’ samples would be better able to act as ‘self-matrix’. Thus, differences in structure and composition of the samples appear to go some way towards explaining differences between trends observed in the presence/absence of matrix. Dependence of MALDI spectra on molecular structure has also been found for two sets of biomass derived samples. Considerably more work appears warranted to determine MALDI-MS parameters appropriate to the analysis of the more oxygenated, lower rank, samples.

Catalytic activity of fullerenes for hydrocracking coal extracts

A liquefaction extract of Point of Ayr coal (UK) was hydrocracked using a mixture of fullerenes as dispersed catalyst and tetralin as solvent. Structural characteristics of the hydrocracking products were examined as a function of increasing reaction time and compared with those of products prepared in the absence of catalyst. Comparison of t.g.a.-derived boiling point distributions showed that the proportion of material boiling at temperatures below 450°C progressively increased with reaction time up to 120 min. Size exclusion chromatograms (s.e.c.) of products indicated a similar steady reduction in the proportion of material observed at the exclusion limit of the column, at up to 90 min reaction. However, reaction between 90 and 120 min appeared to have the reverse effect. Comparison with hydrocracking in the absence of catalyst gave no indication that the fullerenes enhance reductions in boiling point distributions or in apparent molecular masses. MALDI-m.s. largely confirmed findings by s.e.c. The evidence strongly suggests that fullerenes used in these experiments were unable to retain their integrity during the process.