Desorption electrospray ionization-mass spectrometry (DESI-MS) was evaluated for the detection of proteins ranging in molecular weight from 12 KDa to 66 KDa. Proteins were uniformly deposited on a solid surface without pretreatment and analyzed with a DESI source coupled to a quadrupole ion trap mass spectrometer. DESI parameters optimized for protein detection included solvent flow rate, temperature of heated capillary tube, incident and reflection angle, sheath gas pressure, and ESI voltage. Detection limits were obtained for all protein standards and they were found to decrease with decreasing protein molecular weight: for cytochrome-C (12.3 KDa) and lysozyme (14.3 KDa) a detection limit of 4 ng/mm 2 was obtained; for apomyoglobin (16.9 KDa) 20 ng/mm 2 ; for β-lactoglobulin B (18.2 KDa) 50 ng/mm 2 ; and for chymotrypsinogen-A (25.6 KDa) 100 ng/mm 2 . The DESI-MS analysis of higher molecular weight proteins such as ovalbumin (44.4 KDa) and bovine serum albumin (66.4 KDa) yielded mass spectra of low signal-to-noise ratio (S/N), making their detection and molecular weight determination difficult. In this study, DESI-MS proved to be a rapid and robust method for accurate MW determination for proteins up to 17 KDa under ambient conditions. Finally, we demonstrated the DESI-MS detection of the bacteriophage MS 2 capsid protein from crude samples with minimal sample preparation.

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.

A rapid method for identifying specific bacteria from complex biological mixtures using immunomagnetic separation coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been developed. The technique employs commercially available magnetic beads coated with polycolonal antibodies raised against specific bacteria and whole cell analysis by MALDI-MS. A suspension of a bacterial mixture is mixed with the immunomagnetic beads specific for the target microorganism. After a short incubation period (20 mins) the bacteria captured by the beads are washed, resuspended in deionized H(2)O and directly applied onto a MALDI probe. Liquid suspensions containing bacterial mixtures can be screened within 1 h total analysis time. Positive tests result in the production of a fingerprint mass spectrum primarily consisting of protein biomarkers characteristic of the targeted microorganism. Using this procedure, Salmonella choleraesuis was isolated and detected from standard bacterial mixtures and spiked samples of river water, human urine, and chicken blood.

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.

Fatty acid methyl esters (FAMEs) were generated in situ, during pyrolysis, from whole-cell bacterial samples and analyzed by mass spectrometry (MS). The FAME profiles obtained by an in situ thermal hydrolysis methylation (THM) step were compared with gas chromatography (GC) and MS analyses of the chemically extracted and methylated fatty acids. This correlation was based on the ability of each technique to differentiate a representative group of 15 bacteria at the species level as predicted by principal component analysis. All three analyses, GC/FAME, pyrolysis-MS/FAME, and in situ THM-MS/FAME differentiated the studied bacterial sample set into three discrete clusters. The bacteria comprising each cluster were the same for all three analyses, showing that taxonomic information of the lipid profiles was preserved in the Py-MS/FAME and in situ THM-MS/FAME analyses of whole cells. Contributions from saturated, unsaturated, cyclopropyl, and branched bacterial fatty acids to the differentiation of microorganisms were identified for all three analyses. The in situ THM-MS/FAME approach is simple, requires small samples (approximately 2 x 10(6) cells/profile), and is rapid, with a total analysis time under 5 min/sample.