On-probe sample pretreatment for the detection of proteins above 15 KDa from whole cell bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract

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.

MALDI-TOF-MS analysis of bacterial spores: Wet heat-treatment as a new releasing technique for biomarkers and the influence of different experimental parameters and microbiological handling

“…They describe 2,6-dihydroxyacetophenon, which is an alternative for 2,4,6-THAP, as an unsuitable matrix [50]. Similar inconsistent observations are described for the analysis of whole bacterial cells [8,27,28,[51][52][53][54]. Unfortunately, these results are difficult to compare due to strong structural differences between bacterial spores and their vegetative cells or fungal spores.…”

MALDI mass spectrometry analysis of high molecular weight proteins from whole bacterial cells: Pretreatment of samples with surfactants

“…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.…”

“…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.…”

“…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].…”

Applications of MALDI‐TOF Mass Spectrometry in Clinical Diagnostic Microbiology

“…DHB and CHCA are usually optimal for the detection of lower mass ions (Hathout et al, 2000;Williams et al, 2003;Ruelle et al, 2004) with a detection up to 10 kDa when the proper solvent is used. Both SA and FA have been shown to be better for the detection of higher mass ions (above 15 kDa) (Madonna et al, 2000;Conway et al, 2001;Ruelle et al, 2004;Vargha et al, 2006) but provide a lower sensitivity than CHCA (Wang et al, 1998;Ruelle et al, 2004).…”