Assessment of the stability of cell-surface components of microorganisms by MALDI-TOF-MS following preservation on lenticule discs.

Strains representing the species Campylobacter coli, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, and Staphylococcus aureus were randomly selected to assess the consistency of cells preserved on lenticule discs to those archived in traditional freeze-dried ampoules. Each matched pair was cultured using identical conditions and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) to profile the surface-associated molecules of the cells. In addition, the cytosolic/membrane-bound proteins of C. coli and S. aureus strains were further analysed by surface-enhanced laser desorption/ionization time-of-flight MS. The mass spectral profiles in all cases showed a high degree of concordance between cells preserved by both methods and suggest that the properties of cells preserved on lenticule disc are consistent with those archived by the traditional method of freeze-drying.

Matrix-assisted laser desorption ionisation-time-of of-flight mass spectrometry of intact cells allows rapid identification of Burkholderia cepacia complex.

The present study examined the potential of intact-cell matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid identification of Burkholderia cepacia complex (Bcc) bacteria using an Applied Biosystems 4700 Proteomics Analyser. Two software packages were used to analyse mass profiles based on densitometric curves and peak positions. The 75 strains examined, represented the nine established Bcc species and some commonly misidentified species, closely related or biochemically similar to Bcc and relevant in the context of cystic fibrosis microbiology. All Bcc strains clustered together, separated from non-Bcc strains. Within Bcc, most Bcc strains grouped in species specific clusters, except for Burkholderia anthina and Burkholderia pyrrocinia strains which constituted a single cluster. The present study demonstrates that MALDI-TOF MS is a powerful approach for the rapid identification of Bcc bacteria.

Compilation of a MALDI-TOF mass spectral database for the rapid screening and characterisation of bacteria implicated in human infectious diseases.

A database of MALDI-TOF mass spectrometry (MS) profiles has been developed with the aim of establishing a high throughput system for the characterisation of microbes. Several parameters likely to affect the reproducibility of the mass spectrum of a taxon were exhaustively studied. These included such criteria as sample preparation, growth phase, culture conditions, sample storage, mass range of ions, reproducibility between instruments and the methodology prior to database entry. Replicates of 12 spectra per sample were analysed using a 96-well target plate containing central wells for peptide standards to correct against mass drift during analysis. The quality of the data was assessed statistically prior to database addition using root mean squared values of <3.0 as the criterion for rejection. Cluster analysis using a nearest neighbour algorithm also enabled subsets of data to be compared. This was achieved using the bespoke MicrobeLynx trade mark software. Columbia blood agar was used to standardise all procedures for the database, since it permitted the culture of most human pathogens and also produced spectra with a broad range of mass ions. In some instances, alternative media such as CLED were used in specific studies with greater success. Following standardisation of the procedure, a database was developed comprising ca. 3500 spectra with multiple strain entries for most species. The results to date show unequivocally that as the number of strains per species increased, so too did the success of species matching. The technique demonstrated unique mass spectral profiles for each genus/species, with the variation in mass ions among strains/species being dependent on the intra-specific diversity. The success of identification against the database for wild-type strains ranged between 33 and 100%; the lower percentage results being generally associated with poor representation of some species within the database. These findings provide a new dimension for the rapid and high throughput characterisation of human pathogens with potentially broad applications across the field of microbiology.

High throughput identification of clinical isolates of Staphylococcus aureus using MALDI-TOF-MS of intact cells.

Staphylococcus aureus remains an important human pathogen responsible for a high burden of disease in healthcare and community settings. The emergence of multidrug-resistant strains is of increasing concern world-wide. The identification of S. aureus is currently based upon phenotypic and genotypic methods. Here, an alternative approach involving mass spectral analysis of surface-associated proteins of intact bacterial cells by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) was investigated using 95 isolates obtained directly from a clinical laboratory at The Royal London Hospital and 39 isolates from the Staphylococcal Reference Unit, Health Protection Agency, London. Results obtained indicate that clinical isolates share many common mass ions with-type/reference strains which allowed their correct identification when searched against a comprehensive database that has been in the process of development for several years. The existing database contains more than 5000 profiles of various bacterial pathogens, but comprises mainly type or reference strains. The MicrobeLynx software successfully identified all isolates to the correct genus and all but four to the correct species. These were misidentified in the first instance due to contamination or low mass ion intensity but once the cultures were purified and re-analysed they were confirmed as S. aureus by both MALDI-TOF-MS and 16S rRNA sequence analysis. The high percentage of correct identifications coupled with the high speed and the minimal sample preparation required, indicate that MALDI-TOF-MS has the potential to perform high throughput identification of clinical isolates of S. aureus despite the inherent diversity of this species. The method is, however, only reproducible if variable parameters such as sample preparation, media, growth condition, etc. are standardised.

Improved cell typing by charge-state deconvolution of matrix-assisted laser desorption/ionization mass spectra.

Robust, specific, and rapid identification of toxic strains of bacteria and viruses, to guide the mitigation of their adverse health effects and optimum implementation of other response actions, remains a major analytical challenge. This need has driven the development of methods for classification of microorganisms using mass spectrometry, particularly matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), that allows high-throughput analyses with minimum sample preparation. We describe a novel approach to cell typing based on pattern recognition of MALDI mass spectra, which involves charge-state deconvolution in conjunction with a new correlation analysis procedure. The method is applicable to both prokaryotic and eukaryotic cells. Charge-state deconvolution improves the quantitative reproducibility of spectra because multiply charged ions resulting from the same biomarker attaching a different number of protons are recognized and their abundances are combined. This allows a clearer distinction of bacterial strains or of cancerous and normal liver cells. Improved class distinction provided by charge-state deconvolution was demonstrated by cluster spacing on canonical variate score charts and by correlation analyses. Deconvolution may enhance detection of early disease state or therapy progress markers in various tissues analyzed by MALDI-MS.