Application Of Matrix-assisted Laser Research Articles

The Application of Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry to Profile the Surface of Intact Bacterial Cells

Matrix-Assisted Laser Desorption/lonisation Time of Flight Mass Spectrometry (MALDI-TOF-MS) as a tool for differentiating bacterial species was examined using reference strains representing gram-positive and gram-negative taxa. Initially, the effect of differences in medium composition on spectral profile was examined. The results indicated that growth on Columbia blood agar resulted in a larger spectrum of ionized residues and was therefore used for the cultivation of all strains in the rest of the study. The stability of the obtained mass spectral profiles against differences in batch and media processing suggested that no significant alterations to the profiles occurred in response to changes in media sources. The established conditions from these initial experiments were used to standardize subsequent experiments. The MALDI-TOF-MS profiles of 15 reference strains were compared and species characteristic markers were identified. The potential of using MALDI-TOF-MS as a tool for probing clonal diversity was examined using well characterized but clonally variable isolates of Bacteroides fragilis . Comparative analysis of the profiles of 20 strains revealed 5 clusters within the species but compared to other taxa such as Bacteroides merdae and Salmonella arizonae they form a closely related lineage. These results obtained strongly support the potential to use MALDI-TOF-MS as a tool for exploring bacterial surfaces for characteristic biomarkers and species-specific signatures.

Application of matrix-assisted laser desorption/ionization to on-line aerosol time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectra were obtained from single biological aerosol particles using an aerosol time-of-flight mass spectrometer (ATOFMS). The inlet to the ATOFMS was coupled with an evaporation/condensation flow cell that allowed the aerosol to be coated with matrix material as the sampled stream entered the spectrometer. Mass spectra were generated from aerosol composed either of gramicidin-S or erythromycin, two small biological molecules, or from aerosolised spores of Bacillus subtilis var niger. Three different matrices were used: 3-nitrobenzyl alcohol, picolinic acid and sinapinic acid. A spectrum of gramicidin-S was generated from approximately 250 attomoles of material using a molar ratio of 3-nitrobenzyl alcohol to analyte of approximately 20:1. A single peak, located at 1224 Da, was obtained from the bacterial spores. The washing liquid and extract solution from the spores were analyzed using electrospray mass spectrometry and subsequent MS/MS product ion experiments. This independent analysis suggests that the measured species represents part of the B. subtilis peptidoglycan. The on-line addition of matrix allows quasi-real-time chemical analysis of individual, aerodynamically sized particles, with an overall system residence time of less than 5 seconds. These results suggest that a MALDI-ATOFMS can provide nearly real-time identification of biological aerosols. Copyright 2000 John Wiley & Sons, Ltd.

Characterization of fungal spores by laser desorption/ionization time-of-flight mass spectrometry.

A considerable volume of research has now been completed on the application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the analysis of bacteria; however, to date no definitive studies have been made using this technique on fungi. Preliminary studies on the application of the MALDI-MS methodology, previously developed for the analysis of bacteria, to the analysis of intact fungal spores are described here. MALDI-MS and electrospray mass spectrometry enable the high molecular weight analysis of proteins, glycoproteins, oligosaccharides and oligonucleotides. Using MALDI-MS with bacteria has demonstrated the ability to produce 'fingerprints' of the intact cells with the ions observed being associated with the proteinaceous components of the cell wall. This paper reports the adaptation of this technique to the direct analysis of fungal cells. The high percentage of carbohydrate in the fungal cell wall indicates that the ions observed in the mass spectrometric experiments may be of carbohydrate origin. Penicillium spp., Scytalidium dimidiatum and Trichophyton rubrum have been studied in this preliminary investigation and all show individually distinctive spectra which would appear to provide a profile of the cellular material with discrete peaks being observed over the mass range 2 to 13 kDa. The spectra obtained are reproducible within the method used but, as shown in our previous studies on bacteria, washing may selectively release components from the fungal cell wall.

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to insoluble glucose oligomers in decomposed cellulose.

An analytical approach is described for the molecular weight characterization of glucose oligomers from cellulose which had been decomposed by hot-compressed water (HCW). Microcrystalline cellulose was decomposed to 18% of water insoluble fraction (WI) plus 82% of water soluble fraction (WS) by HCW (295 degrees C, 10 mL/min). The glucose oligomers in the WI were analyzed by pericullar anion exchange chromatography, and oligomers longer than pentamers were detected. These results were also confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Many peaks derived from glucose oligomers with molecular weights less than 4000 Da were detected in the spectra. Mass differences between peaks were 162 Da, as expected for polymers composed of glucose units. This appears to be the first report concerning molecular weight determination of water or organic solvent insoluble compounds like glucose oligomers from cellulose using MALDI-TOFMS.

The Application of Matrix-Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry to Profile the Surface of Intact

Matrix-Assisted Laser Desorption:lonisation Time of Flight Mass Spectrometry (MALDI-TOF-MS) as a tool for differentiating bacterial species was examined using reference strains representing gram-positive and gram-negative taxa. Initially, the effect of differences in medium composition on spectral profile was examined. The results indicated that growth on Columbia blood agar resulted in a larger spectrum of ionized residues and was therefore used for the cultivation of all strains in the rest of the study. The stability of the obtained mass spectral profiles against differences in batch and media processing suggested that no significant alterations to the profiles occurred in response to changes in media sources. The established conditions from these initial experiments were used to standardize subsequent experiments. The MALDI-TOF-MS profiles of 15 reference strains were compared and species characteristic markers were identified. The potential of using MALDI-TOF-MS as a tool for probing clonal diversity...

Structural Analysis of Cartilage Proteoglycans and Glycoproteins Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Cartilage extracellular matrix molecules synthesized and maintained by chondrocytes form a strong, elastic tissue functioning to cushion and protect the subchondral bone. Osteoarthritis is characterized by degradation of cartilage extracellular matrix molecules resulting in fibrillation, irreversible erosion, and eventual failure of the tissue. With recent interest in the degradation of cartilage extracellular matrix molecules, a need for more detailed structural information exists. Posttranslational modifications are believed to play a role in determining the susceptibility of these molecules to proteolytic degradation during the development of osteoarthritis. The purpose of this paper is to show how the application of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to extracellular matrix protein and proteoglycan structure will help elucidate problems in extracellular matrix biochemistry. Methodological issues relating to the high molecular weight, polydispersity, and high degree of posttranslational modification of these molecules are discussed. MALDI-TOF mass spectrometry provides an improved level of detail for extracellular matrix protein and proteoglycan structure and is useful in addressing issues surrounding the causes of degradation during osteoarthritis.

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to the structure determination of medium and large macrocycles formed by palladium(0)-catalyzed allylation of arenesulfonamides, sulfamide, and cyanamide.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry allowed the direct determination of the extent of macrocyclic and linear oligomer formation in the palladium(0)-catalyzed allylation of highly acidic and non-nucleophilic arenesulfonamides, sulfamide, and cyanamide. Palladium-containing 15-membered-ring macrocyclic compounds gave unusual [M - H](+) ions besides [M + Na](+) and [M + K](+) adducts. Copyright 1999 John Wiley & Sons, Ltd.

Application of matrix-assisted laser desorption/ionization mass spectrometry to the detection of melanins formed from Dopa and dopamine.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to study melanogenesis starting from Dopa and dopamine, the latter considered one of the precursors of neuromelanins. These substrates were left to react with the peroxidase - H(2)O(2) system, which is postulated to play an important role in melanin biosynthesis. Samples were prepared by ultrafiltering the substrate - enzyme solution after 30, 60, 120, 240 and 360 min of reaction and aliquots were immediately lyophilized. The reaction of dopamine with peroxidase - H(2)O(2) favoured the formation of dopamine oligomers up to octamers. In contrast, the action of either peroxidase or H(2)O(2) alone, studied for comparison, did not lead to melanin production and only dimeric and trimeric species were observed. Also for Dopa, analogous results were obtained in the presence of either peroxidase or H(2)O(2) alone, without melanin formation. Conversely, Dopa with the peroxidase - H(2)O(2) system led to the formation of a black precipitate after 120 min of reaction, and oligomers of 5,6-dihydroxyindole (DHI), an intermediate of melanogenesis, were detected, together with products of further oxidation. Faster kinetics were observed when Dopa was treated with tyrosinase, the enzyme catalysing the oligomerization of tyrosine to melanins, leading to the formation mainly of DHI oligomers.

Rapid semi-on-line monitoring of Fmoc solid-phase peptide synthesis by matrix-assisted laser desorption/ionization mass spectrometry

A simple yet highly effective application of matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS) for the rapid monitoring of Fmoc solid-phase peptide synthesisis described. A few beads of the resin are removed at any desired step during synthesis, thefully protected peptide is cleaved from the resin and an MS spectrum of the analytes presentis produced. Some standard side-chain protecting groups may be cleaved off during samplepreparation for MS analysis; however, these cleavages are readily identified. Using thisapproach, incomplete amino acid acylations are readily detected in approximately the sametime as by traditional tests such as ninhydrin. The semi-on-line method also lends itself toready optimization of synthesis protocols and to the examination of resin-bound peptide sidereactions which may not be detectable by chemical means.

Application of matrix-assisted laser desorption/ionization Fourier transform mass spectrometry to the analysis of planar porphyrins and highly substituted nonplanar porphyrins

The applicability of matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry to the analysis of porphyrins has been examined. High resolution spectra were readily obtained with good sensitivity and a detection limit as low as 2 fmol. A mixed solvent system of toluene and ethanol (1 : 1 by volume) proved to be compatible with the 2,5-dihydroxybenzoic acid (DHB) matrix and solubilized most of the porphyrins examined in this study. Porphyrins which were insoluble in this solvent mixture could be dissolved in an appropriate solvent and deposited on a layer of DHB (layered MALDI). The parent ion was generally the largest peak in the spectrum, although for some metalloporphyrins the peak corresponding to (M ‐ metal + 3H) + was dominant. The extent of demetallation was found to depend on factors such as sample preparation, the metal ion and the laser intensity. Addition of more than one hydrogen occurred in many cases but was a minor process. Spectra of compounds ionized by different methods showed increasing fragmentation in the order MALDI < layered MALDI < fast-atom bombardment < laser desorption.

The Application of Matrix-assisted Laser Desorption/Ionization Combined with Collision-induced Dissociation to the Analysis of Synthetic Polymers

Matrix-assisted laser desorption/ionization combined with collision-induced dissociation (CID) has been applied to the structural determination of synthetic polymers. Post-source decay/CID experiments on a time-of-flight (TOF) instrument have been compared with CID data from a hybrid sector-TOF mass spectrometer. Fragment-ation spectra of polymers, with molecular weights of up to 4500 Da, have been shown to aid structural and end-group determination. The polymers studied were poly(methyl methacrylate), poly(ethylene glycol) and poly(ethylene terephthalate).