ornamentals. Since the last century, outbreaks of this disease have caused economical crisis in agriculture [10]. Results We describe a standardized sample preparation and analytical procedure for easy bacterial classification and identification by MALDI mass spectrometry detection of protein mass patterns (Figure 1A). This method includes the use of advanced bioinfor- matics analysis and a database resource containing a comprehen- sive number of bacterial reference mass spectra. We enlarged the potential of this approach by genotyping an informative single nucleotide polymorphism (SNP) by mass spectrometry. All Erwinia bacteria analyzed in this study were conventionally cultured in liquid medium. To fulfill biological safety requirements the potentially pathogenic Erwinia samples were inactivated efficiently in ,73% ethanol. This treatment completely destroyed the viability of the bacteria after an hour of fixation. Once the samples were fixed, they were treated with formic acid for cell wall disruption and acetonitrile for protein extraction (Materials and Methods). Finally, a fraction of the protein samples was prepared on a MALDI target plate and mass spectra accumulation was performed automatically. Applying the standardized experimental procedure including culturing in liquid media (Materials and Methods), we generated a reference mass spectra database containing main spectra libraries (MSPs) of bacteria of the genus Erwinia and some other related Bacteria/Mass Spectrometry bacteria. Therefore, we accumulated twenty mass spectra to achieve above average quality spectra. Protein mass patterns were detected in the mass range of 2,000 to 20,000 Da. The building of a general database of reference mass spectra, which are produced by the standardized protocol applied in this study, is currently underway. To date, this database comprises more than 2800 bacterial strains, including the genera Pantoea, Shigella, Listeria, Salmonella, and Klebsiella (Materials and Methods). The database has been implemented in our analysis software (Materials and Methods) and was used for the identification experiments shown below. To reproduce the results of this study and to test the software for additional applications, the software package and the reference mass spectra are freely available as a CD that can be requested from the authors. For phylogenetic analysis, we clustered hierarchically mass spectra of type strains and others in dendrograms according to their mass signals and intensities (Figure 1B). Each reference spectrum of a dataset was compared with the other reference spectra, thus resulting in a matrix of cross-wise identification values. This matrix is used to calculate the distance values for each pair. Based on these distance values the dendrogram was generated using the according function of the statistical toolbox of Matlab 7.1 (The MathWorks Inc., USA), which was integrated in the analysis software (Materials and Methods). The clustering approach applied was based on similarity scores implemented in the analysis software. For comparison, a phylogenetic dendrogram was generated from 16S rRNA sequences (Figure 2). Interestingly, Figure 1. A: A general scheme of the procedure. Bacterial colonies are subjected to chemical treatment. Samples can be analyzed within a few minutes by MALDI mass spectrometry and mass spectra are transferred to analysis and identification software. B: Classification of bacteria. Based on the protein mass patterns, bacterial strains can be clustered hierarchically. A dendrogram generated by this approach including a comprehensive set of Erwinia type strains was displayed. Species with distance levels over 500 had completely different mass signal patterns. Comparison of spectra of these species for a distance measure was thus uninformative. Strains clustering with distance levels lower than 500 could be classified up to the species and partially to subspecies level. The limit of resolution was set by the distances derived from measurement variability. doi:10.1371/journal.pone.0002843.g001 PLoS ONE | www.plosone.org 2 July 2008 | Volume 3 | Issue 7 | e2843