Discrimination of different species from the genus Drosophila by intact protein profiling using matrix-assisted laser desorption ionization mass spectrometry

Abstract

Background: The use of molecular biology-based methods for species identification and establishing phylogenetic relationships has supplanted traditional methods relying on morphological characteristics. While PCR-based methods are now the commonly accepted gold standards for these types of analysis, relatively high costs, time-consuming assay development or the need for a priori information about species-specific sequences constitute major limitations. In the present study, we explored the possibility to differentiate between 13 different species from the genus Drosophila via a molecular proteomic approach.

Results: After establishing a simple protein extraction procedure and performing matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) with intact proteins and peptides, we could show that most of the species investigated reproducibly yielded mass spectra that were adequate for species classification. Furthermore, a dendrogram generated by cluster analysis of total protein patterns agrees reasonably well with established phylogenetic relationships.

Conclusion: Considering the intra- and interspecies similarities and differences between spectra obtained for specimens of closely related Drosophila species, we estimate that species typing of insects and possibly other multicellular organisms by intact protein profiling (IPP) can be established successfully for species that diverged from a common ancestor about 3 million years ago.

Figure 1

Comparison of six MALDI-MS spectra, 3.3 to 4.6 kDa. The upper three spectra (red) were obtained with three different flies from D. ananassae, whereas the lower three (blue) were from different (female) D. melanogaster specimens. Peaks detected by FlexAnalysis are indicated for two representative spectra. Peaks highlighted in green were used for stringent species discrimination in the generation of the final dendrogram (Fig. 4).

Figure 2

Results from unbiased cluster analysis. 125 spectra from flies of 13 different species were clustered using Dice coefficients. A color (heatmap) representation of the pairwise similarity (Dice) coefficients is shown in the lower part of the figure, the white square outlining the cluster of D. mauritiana spectra. Above this, clustering of individual spectra, color-coded according to species, are shown in a dendrogram. Percentages of bootstrapping replicates supporting the location of individual nodes are indicated. On top of this, a second dendrogram indicating the phylogeny of the different species is shown.

Figure 3

Scatterplots from unbiased PCAs using 125 spectra from flies of 13 different species. Species-specific color-coding corresponds to that shown in Fig. 2. Spectra belonging to one species are outlined by a convex shape or an ellipse. A: PCA containing all spectra, with 95% concentration ellipses for three subgenera/species groups. B: PCA using only spectra from D. pseudoobscura and D. miranda, with 50% concentration ellipses. C: PCA performed with spectra from the subgenus Drosophila but without D. pseudoobscura and D. miranda, with 95% concentration ellipses. D: PCA of the spectra from the subgenus Sophophora, with 95% concentration ellipses.

Figure 4

Results from cluster analysis using 208 selected peaks. For further information, please refer to the legend of Fig. 2.

Figure 5

Putative PEB2 from D. melanogaster at 4977.7 Da. Ten spectra shown in green were obtained from male specimens, red spectra derived from 10 female flies.