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

Abstract

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).

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.…”

Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification

“…The spectral repeatability observed here compared to the concerns over the MALDI-MS analysis of whole bacterial cells [14,17,20,21] and the sensitivity achievable, reportedly an order of magnitude better compared to MALDI-MS [15], suggests that ESI-MS can be advantageous over MALDI-MS as well as complementary to it for microbial characterization. …”

“…The solvent (aqueous acidic acetonitrile) in which the bacteria were suspended in the present study is known to assist the ESI process, has been commonly employed for MS of biomacromolecules [46,47], and has been used in LC-ESI-MS [31] and MALDI-MS investigations [15,20,48,49] for microbial characterization. However, little is known about the effect of solvent composition on the extraction process and in turn the spectral information.…”

“…For instance, the dried sample matrix preparation is inhomogeneous, requiring the selection of "sweet" spots on the target that would give the best ion formation, and matrix adduct ions are often formed that decrease effective mass resolution and decrease the possibility of analyzing low (m/z Ͻ 1000) molecular weight species. Moreover, there are still concerns of spectral reproducibility, owing to the sample preparation steps [14,17,20,21], although some investigations [19,22,23] show that this problem can be overcome.…”

Rapid identification of stress-related fingerprint from whole bacterial cells of Bifidobacterium lactis using matrix assisted laser desorption/ionization mass spectrometry

“…MALDI-TOFMS has been used during the past years for the direct determination of proteins in a complex biological mixture without prior separation at high sensitivity levels (i.e., low femtomoles). In particular, MALDI mass spectrometry has been described for generating marker profiles directly from unfractionated micro-organisms such as viruses, bacteria, fungus cells, and spores [2][3][4][5][6][7][8][9][10][11]. This approach enables detection, identification and characterization of peptides and proteins from "intact" micro-organisms and was applied in various fields such as biotechnology, cell biology, and pharmaceutical research [7].…”