Species determination of Culicoides biting midges via peptide profiling using matrix-assisted laser desorption ionization mass spectrometry

Abstract

Background: Culicoides biting midges are vectors of bluetongue and Schmallenberg viruses that inflict large-scale disease epidemics in ruminant livestock in Europe. Methods based on morphological characteristics and sequencing of genetic markers are most commonly employed to differentiate Culicoides to species level. Proteomic methods, however, are also increasingly being used as an alternative method of identification. These techniques have the potential to be rapid and may also offer advantages over DNA-based techniques. The aim of this proof-of-principle study was to develop a simple MALDI-MS based method to differentiate Culicoides from different species by peptide patterns with the additional option of identifying discriminating peptides.

Methods: Proteins extracted from 7 Culicoides species were digested and resulting peptides purified. Peptide mass fingerprint (PMF) spectra were recorded using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and peak patterns analysed in R using the MALDIquant R package. Additionally, offline liquid chromatography (LC) MALDI-TOF tandem mass spectrometry (MS/MS) was applied to determine the identity of peptide peaks in one exemplary MALDI spectrum obtained using an unfractionated extract.

Results: We showed that the majority of Culicoides species yielded reproducible mass spectra with peak patterns that were suitable for classification. The dendrogram obtained by MS showed tentative similarities to a dendrogram generated from cytochrome oxidase I (COX1) sequences. Using offline LC-MALDI-TOF-MS/MS we determined the identity of 28 peptide peaks observed in one MALDI spectrum in a mass range from 1.1 to 3.1 kDa. All identified peptides were identical to other dipteran species and derived from one of five highly abundant proteins due to an absence of available Culicoides data.

Conclusion: Shotgun mass mapping by MALDI-TOF-MS has been shown to be compatible with morphological and genetic identification of specimens. Furthermore, the method performs at least as well as an alternative approach based on MS spectra of intact proteins, thus establishing the procedure as a method in its own right, with the additional option of concurrently using the same samples in other MS-based applications for protein identifications. The future availability of genomic information for different Culicoides species may enable a more stringent peptide detection based on Culicoides-specific sequence information.

Figure 1

Results from cluster analysis. 64 spectra from biting midges of 7 different species were clustered using Dice coefficients. A colour (heatmap) representation of the pairwise similarity (Dice) coefficients is shown. Above this, clustering of individual spectra, color-coded according to species, are shown in a dendrogram. On the left side, the same dendrogram is shown but color-coded according to species group.

Figure 2

Comparison of PCR dendrogram (A) and MS dendrogram (B). A: The PCR dendrogram is based on the COX1 DNA sequencing data, suggesting a possible phylogeny for the different species. B: The MS dendrogram is based on the MALDI-TOF MS data. Percentages of bootstrapping replicates supporting the location of individual nodes are indicated.

Figure 3

Scatterplot from unbiased PCAs using 64 spectra from midges of 7 different Culicoides species. Species-specific colour coding corresponds to that shown in Figure 1. Spectra belonging to one species are outlined by convex shape. The dashed lines indicate 95% concentration ellipses. A: PCA containing all spectra; B spectra from the obsoletus group only; C: spectra from the pulicaris group only.

Figure 4

The top 40 ranked peaks and their corresponding CAT scores of the SDA analysis for Culicoides species (A) and species groups (B). With highest ranking peaks near the top of the table, the length and direction of the horizontal blue bars indicate the CAT scores of the centroid versus the pooled mean and as such describe the influence of a certain peak in differentiating between Culicoides species or species groups. For example, the top-ranking peak in A contributes strongly to the separation of C. nubeculosus from all other species, as highlighted by the length of the bar in the respective column (large positive CAT score) and the opposite direction of the bars in the columns from the bars of the other species (negative CAT scores).

Figure 5

Sections of 7 representative MALDI-TOF MS spectra of the 7 Culicoides species. A: The vertical, dash-dotted lines marked with an asterisk indicate monoisotopic peaks that are characteristic (but not exclusive) for one species (top 3 for each species). Likewise, the vertical, dashed lines marked with a triangle denote monoisotopic peaks that are characteristic for one certain species group (top 3 for each group). B: Zooms for six exemplary peaks. Except for the peak at 2,253.260 Da the peaks shown are all ranked under the top 40 shown in Figure 4.

Figure 6

MALDI-TOF MS spectrum of a representative specimen of C. punctatus . Peaks representing the peptides of five proteins identified by offline nano-HPLC-MALDI MS/MS are indicated. For better illustration, the m/z range from 2,100 to 2,280 Da has been magnified. x = actin; â–³ = ATP-Synthase alpha subunit (mitochondrial); â–½ = ATP-Synthase beta subunit (mitochondrial); ○ = myosin; □ = tropomyosin.

Figure 7

IPP spectra vs. SMM spectra of C. obsoletus . A: Complete IPP spectrum of a C. nubeculosus specimen from m/z 1.6-16 kDa with ca. 200 detectable peaks. B: A zoom of the m/z range from 1,600-4,020 Da of the IPP spectrum is depicted. This corresponds to the range where the IPP and SMM spectra overlap, ca. 100 peaks are detectable. C: The same range shown for an SMM spectrum of a specimen of the same species; ca. 260 peaks are detectable.