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A rapid methodology is described for the enhancement of the signal‐to‐base‐line (S/B) ratio of high molecular weight protein signals from whole cell bacteria analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS). The procedure involves depositing growing bacteria colonies from culture dishes directly onto the MALDI probe followed by treatment of the sample spot with a 2 µL aliquot of 40% ethanol prior to the addition of a ferulic acid matrix solution (12.5 mg dissolved in 17% formic acid/33% acetonitrile/50% H2O). Protein signals of more than 20 kDa were routinely produced from both Gram positive and Gram negative bacteria prepared in this manner. Moreover, a substantial number of intense protein signals were also produced in the more ‘conventional’ fingerprint region extending from 4 to 20 kDa. This approach is rapid, easy to implement into existing methodologies, and does not require any special hardware. Copyright © 2000 John Wiley & Sons, Ltd.

Desorption electrospray ionization (DESI) mass spectrometry (MS) was used to differentiate seven bacteria species on the basis of their measured DESI-mass spectral profile. Both gram-positive and gram-negative bacteria were tested and included Escherichia coli, Staphyloccocus aureus, Enterococcus sp., Bordetella bronchiseptica, Bacillus thuringiensis, Bacillus subtilis and Salmonella typhimurium. Distinct DESI-mass spectra, in the mass range of 50-500 u, were obtained from whole bacteria in either positive or negative ion modes in less than 2 mins analysis time. Positive ion DESI-mass spectral fingerprints were compared using principal components analysis (PCA) to investigate reproducibility for the intraday and the day-to-day measurements and the method selectivity to differentiate the bacteria studied. Detailed study of variances in the assay revealed that a large contribution to the DESI-mass spectral fingerprint variation was the growth media preparation procedure. Specifically, experiments conducted with the growth media prepared using the same batch yielded highly reproducible DESI-mass spectra, both in intraday and in day-to-day analyses (i.e. one batch of growth media used over a 3-day period versus a new batch every day over the same 3-day period). Conclusions are drawn from our findings in terms of strategies for rapid biodetection with DESI-MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), utilizing an on-probe sample pretreatment, was applied to the rapid and direct detection of intact phospholipids from whole bacterial cells. The sample preparation procedure involved depositing growing bacterial colonies from culture dishes directly onto the MALDI probe followed by treatment of the sample spot with a 3 micro L aliquot of an aqueous 0.05 M solution of sodium iodide prior to the addition of a 2,5-dihydroxybenzoic acid (DHB) matrix solution (ca. 8 mg dissolved in 70% acetonitrile/30% H(2)O containing 0.1% of trifluoroacetic acid). The MALDI spectra obtained from whole bacteria cells showed a series of ions generated from bacterial phospholipids, such as phosphatidylethanol-amines (PEs) and phosphatidylglycerols (PGs), which were clearly observed as well-resolved peaks. The ranges of the observed total carbon numbers in two acyl groups for PEs and PGs (30-36 and 33-36, respectively) were in good agreement with those reported previously. Furthermore, the distinct discrimination of four species of the Enterobacteriaceae family cultured identically was achieved by using principal components analysis (PCA) conducted on the relative peak intensities of phospholipids observed from the MALDI spectra.

BackgroundEnzyme based remediation of wastewater is emerging as a novel, efficient and environmentally-friendlier approach. However, studies showing detailed mechanisms of enzyme mediated degradation of organic pollutants are not widely published.ResultsThe present report describes a detailed study on the use of Soybean Peroxidase to efficiently degrade Trypan Blue, a diazo dye. In addition to examining various parameters that can affect the dye degradation ability of the enzyme, such as enzyme and H2O2 concentration, reaction pH and temperature, we carried out a detailed mechanistic study of Trypan Blue degradation. HPLC-DAD and LC-MS/MS studies were carried out to confirm dye degradation and analyze the intermediate metabolites and develop a detailed mechanistic dye degradation pathway.ConclusionWe report that Soybean peroxidase causes Trypan Blue degradation via symmetrical azo bond cleavage and subsequent radical-initiated ring opening of the metabolites. Interestingly, our results also show that no high molecular weight polymers were produced during the peroxidase-H2O2 mediated degradation of the phenolic Trypan Blue.

The use of surfactants as additives in conjunction with on-probe whole cell bacterial protein analysis employing MALDI-TOF-MS is described. Nonionic and zwitterionic surfactants were used to enhance the detection of high molecular weight proteins. Three nonionic, N-octyl-B-D-glactopyranoside, N-decyl-B-D-maltopyranoside, and N-dodecyl-B-D-maltoside, and two zwitterionic surfactants, N,N-dimethyldodecylamine-N- has been shown to be a powerful tool for the detection of bacterial proteins [1][2][3]. Sample preparation for the bacterial proteins for the MALDI-TOF-MS analysis has ranged from separation using HPLC and two-dimensional polyacrylamide gel electrophoresis (2D PAGE) [4,5] to direct analysis of whole bacterial cells without preseparation [6,7]. Holland et al. first reported on the successful analysis of five individual microorganisms by simply mixing the bacteria with ␣-cyano-4-hydroxycinnamic acid matrix on the MALDI probe followed by MALDI-TOF-MS analysis [6]. Using this approach, bacterial protein profiling was simple and rapid (time Ͻ5 min). A majority of the early research published on whole cell bacterial MALDI analysis described protein profiles up to 30 kDa [2,3]. The signals in this range were attributed to cellular proteins or cell wall associated proteins [8,9].Numerous studies were reported to expand the upper mass range for bacterial proteins obtained from whole cell MALDI-TOF-MS analysis [7,9]. The influence of matrix, solvent, and spotting techniques on the mass range and the quality of the MALDI spectra were reported.The addition of detergents has a positive effect on sample preparation of bacterial lysates for 2D PAGE.