the mass spectrometry and the sequence-based dendrograms showed similar clustering within related species. As for the other cell parameters, co-evolution of 16S rRNA sequences and ribosomal proteins, which are mainly detected by mass spectrom- etry from whole bacterial cells, could be assumed [11]. Thus, the mass spectrometry-based dendrogram made significant sense from a biological point of view. As for 16S rRNA, related species such as E. amylovora and E. pyrifoliae from Korea and Japan or epiphytic bacteria like E. persicina and E. rhapontici clustered closely together. This observation was confirmed by data generated from housekeeping genes, as well as microbiological and biochemical studies [12]. E. tasmaniensis was linked to E. amylovora and to E. pyrifoliae, whereas E. billingiae was placed on a different branch. The two exemplary E. tasmaniensis strains from different isolation origins clustered closely together and their distance based on comparison of protein mass patterns were within the range of experimental noise of the procedure (Figure 1B). The average reproducibility of the procedure is exemplarily documented in Figure 3. In general, we observed coefficient of variation (CV) values slightly above 0.3 in intra- and inter-run experiments. With the entire reference spectra library, we could identify unambiguously bacteria of the genus Erwinia. Therefore, we analyzed a number of isolates from different locations of the world and samples from necrotic wood of diseased pear trees from Carinthia. In many cases, plant samples contained mixtures of bacteria of the genus Erwinia or other genera. Conventional culturing on agar and/or in liquid media prior to chemical treatment was performed to produce sufficient amount and homogeneity of bacterial samples for the analysis. The approach became robust against growth times once the bacteria have entered the stationary phase (data not shown). For initial species Bacteria/Mass Spectrometry identification of the different isolates listed in Table 1, we applied a pattern-matching algorithm, which calculated calibrated m/z values, average intensities, and frequency distributions of each mass signal in different measurements. With our approach, an identification score of 2.0 or higher indicated a reliable identification of species. As summarized in Table 1, we identified unambiguously pathogenic bacteria such as E. rhapontici, E. persicina, E. amylovora, and Brenneria quercinia (syn. Erwinia quercinia) from a variety of plant samples. Figure 4 shows a typical result of an identification experiment performed in this study. Although the bacteria were grown on different media, similar identification scores were obtained due to their almost identical mass spectra (Table 1). We could even detect the fire blight pathogen in washes of plant tissue from in vitro propagated pears that were infected with about 107 cells of E. amylovora. In a larger study, we additionally analyzed over several months a comprehensive number of isolates (Table 1). We screened successfully the mass spectra of these isolates with the entire reference spectra database. In all cases we correctly detected the respective samples with similar identification scores. All mass spectrometry-based identi- fication results presented in this paper were consistent with 16S rRNA sequencing and microbiological and biochemical data derived from intensively studied isolates that were stored in our laboratory. For accurate identification of closely related species such as the plant pathogens E. amylovora and E. pyrifoliae, we used a weighted pattern-matching algorithm. This algorithm uses selected charac- teristic mass signals to which specific values can be assigned in the analysis (Figure 5). As shown in Table 2, weighted pattern- matching helped to neatly determine very closely related strains that could not be distinguished by the initial pattern-matching Figure 2. Phylogenetic analysis. For comparison to mass spectrometry based clustering a conventional 16S rRNA sequence-based dendrogram generated with ClustalW and Mega 3.1 is shown. Phylogenetic distances were estimated by the method of Jukes and Cantor [15]. The tree topology was inferred by neighbor-joining method with a bootstrap value of 1000. For the reconstruction of phylogeny, the neighbor-joining and maximum- parsimony procedures produced similar results. The 16S rRNA sequences of E. coli strains were identical to the E. coli strains shown in the dendrogram generated on the basis of mass spectra. doi:10.1371/journal.pone.0002843.g002 PLoS ONE | www.plosone.org 3 July 2008 | Volume 3 | Issue 7 | e2843