A novel and rapid approach to yeast differentiation using matrix-assisted laser desorption / ionisation time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) was investigated as a method for the rapid identification of yeast cells. Following pretreatment of yeast samples with a cell wall digesting enzyme (lyticase), distinct and reproducible mass spectra over the m/z range 2,000 to 16,000 were obtained by MALDI-TOFMS. Using an optimised procedure, characteristic mass spectra that distinguished between Candida spp. and between strains of Saccharomyces cerevisiae were produced. The approach offers the potential for rapid differentiation of yeasts in clinical diagnosis and in the fermentation industries.


Introduction
There is a need for rapid and accurate methods of yeast differentiation in clinical diagnosis and in the beverage industries.Morphological or biochemical parameters have traditionally been used to distinguish yeast species and individual strains [4].These methods, however, are time consuming and often unreliable.Yeasts are currently identified by molecular biological methods, such as restriction fragment length polymorphism analysis (RFLP) [14] and techniques based on polymerase chain reaction (PCR) technology, for example random amplification of polymorphic DNA (RAPD) [17,21].These methods are slow, labour-intensive and require considerable technical expertise.Several processing steps involving expensive reagents are involved, any one of which can introduce errors that invalidate the assay.For the brewing industry, a further problem has been the need to obtain high-purity DNA from yeasts since fermentation cultures contain inhibitors that influence the activity of Taq polymerase [16].There exists a need therefore to develop more rapid and simplified methods for the accurate differentiation and identification of yeasts.With recent developments in analytical instrumentation, whole-organism fingerprinting by physicochemical spectroscopic methods has become possible.Pyrolysis-mass spectrometry (Py-MS), UV resonance Raman spectroscopy and Fourier transform-infrared spectroscopy (FT-IR) are the most common techniques used for this application [5,15,18].Cumbersome data analysis and the need for skilled interpretation of spectra are general limitations of the whole-organism fingerprinting approach for rapid identification of microorganisms.When applied to differentiation of Candida spp.for example, Py-MS and FT-IR spectra showed very little qualitative difference between species and analysis by multivariate statistical methods was necessary to differentiate species [18].
Matrix Assisted Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) is emerging as a powerful tool for identification of bacteria [19,12].Intact bacterial cells analysed by MALDI-TOF-MS have been shown to give reproducible and repeatable spectra that are simple to interpret visually.The technique detects differences in cell wall constitution through ionisation of surface molecules.Identification of an unknown organism is then based on assignment of genus, species and/or strain specific biomarkers.Intact cell MALDI-TOF-MS has been successfully used for the rapid identification of bacterial cells [1,3,6,7,13].Although this approach has recently been used to characterise fungal spores [20] and filamentous fungi [2], its application to the differentiation and identification of genera, species and strains of yeasts has not been demonstrated.
In the work reported on here, we investigated the possibility of differentiating yeasts by intact cell MALDI-TOF-MS.The aim of the work was to develop a method for generating genus, species or strain specific biomarkers from yeast cell wall components.

MALDI-TOF-MS analysis
Positive ion spectra were acquired with a Lasermat 2000 linear time-of-flight mass spectrometer (Finnegan Mat Ltd., Hemel Hempstead, UK) with an acceleration voltage of 20 kV.Ionisation of molecules was induced using a nitrogen laser at a wavelength of 337 nm.Mass spectra were averaged over 12-24 individual laser shots using dedicated Lasermat 2000 software.Averaged spectra were baseline corrected and smoothed using the proprietary software.

Results and discussion
Initial experiments focused on obtaining characteristic MALDI-TOF-MS spectra from yeast cells in the absence of enzymatic pretreatment of cells.The experimental parameters investigated were matrix chemical, matrix solvent and the ratio of sample to matrix.Distinct peaks were not generated by MALDI-TOF-MS analysis of untreated yeast cells under the range of conditions used.
Many yeast cell wall proteins are covalently bound to glucan and chitin in the cell wall.They are therefore more securely attached to the yeast cell wall than are bacterial cell wall proteins [18].Pretreatment of yeast samples with lyticase (a β1, 3-glucanase preparation) was introduced into the method in an attempt to partially digest the glucan cell wall leading to release cell wall components that may then be ionised by MALDI.Following lyticase treatment of yeast samples, distinct peaks were present in positive ion spectra from MALDI-TOF-MS analysis that were not present in those from untreated cells nor in those from no-cell controls (Figs 1 and 2).These spectra indicate that lyticase treatment of samples gave rise to ionisable molecules from yeast cells that could be detected by MALDI-TOF-MS.The lyticase treatment of yeast samples was optimised by adjusting enzyme concentration and time of treatment.Ionisation of molecules by MALDI was optimised through (1) altering the matrix, (2) matrix solvent and (3) ratio of sample to matrix, as described above.The optimised procedure most suited to generation of distinct peaks in MALDI-TOF-MS analysis of lyticase treated yeast cells was: 100 units lyticase treatment for 15 minutes; 3,5-dimethoxy-4-hydrocinnamic acid as matrix in an aqueous solvent containing trifluoroacetic acid (0.067%) and acetonitrile (33%); sample to matrix ratio of 1 : 9 to 1 : 12 (v/v).
Using the optimised procedure, distinct peaks ranging from m/z 2000 to 16 000 were obtained from each of the yeasts.Analysis of replicate cultures of Saccharomyces cerevisiae NCYC 22118 (Fig. 3) and Candida albicans NCYC 597 (Fig. 4) gave simple and consistent mass spectral profiles within this range.Comparison between different strains of S. cerevisiae (Fig. 5) and different Candida spp.(Fig. 6) showed that a distinctive mass spectral profile was generated for each of the yeasts analysed, which may be used for the purposes of identification.For example, S. cerevisiae NCYC 19201 displayed a strong multiplet of mass ions in the range m/z 2400 to 3400 and a highest mass ion at m/z 6700.Conversely, S. cerevisiae NCYC 22118 displayed only one high-mass ion in the m/z 2400-3400 range (the peak at m/z 3,195 was common to all three S. cerevisiae strains), but released ions detectable at m/z values of 8800-8900 and 11 700.Similarly, the Candida species all released ions with approximate m/z values of 3700 and 7000-7400, whilst also releasing ions specific to the individual species under analysis.
As has been reported for fungal spores [20], the spectra generated by analysis of lyticase-treated yeast cells displayed greater peak width than those generated for bacteria (e.g., [1,3,12]).This may be related to the properties of the yeast cell wall and to the nature of enzymatic pretreatment.Cell wall proteins are covalently linked to β-1,6-glucan, which is in turn linked to β-1,3-glucan and to a lesser extent to chitin [8][9][10][11].Since lyticase is a β-1,3-glucanase with residual β-1,6-glucanase (due to the use of a partially purified enzyme), the majority of proteins exposed to ionisation will be β-1,6-glycosylated [8,9,11].Fractionation of yeast cell wall proteins following β-1,3-glucanase digestion has been reported to yield large heterogeneous molecules, whereas relatively discrete molecules are obtained following digestion with β-1,6-glucanase [8].Our future work will investigate the influence of other yeast cell wall digesting enzymes and enzyme combinations on the generation of species and strain specific biomarkers by MALDI-TOF-MS.

Conclusion
We have shown that following enzymatic treatment of yeast samples characteristic and reproducible spectra can be generated by MALDI-TOF-MS.The analysis can distinguish between Candida spp.and between strains of S. cerevisiae, offering the potential for rapid differentiation and identification of yeasts in clinical diagnosis and in the fermentation industries.