Spectral library
Spectral libraries are essential for effective post-acquisition processing of SWATH data because they contain spectrometric data for all peptide precursors and their respective ion fragments, which are extracted from prior DDA MS experiments [27].
In the current work, we have generated a spectral library for O. moubata saliva proteins from DDA MS experiments involving 3 female and 3 male saliva samples. Additional file 2: Table S1 is the Protein Pilot report showing the spectrometric, statistic and identification data in the spectral library generated from O. moubata saliva proteins. This library included 5,497 spectra associated with 99% confidence, corresponding to 3,734 distinct peptides and 388 protein sequences with a FDR ≤ 1%. After eliminating, from the 388-protein list, up to 65 redundant identifications and 64 hits to non-annotated sequences from the O. moubata sialotranscriptome database, we obtained a final list of 259 non-redundant salivary proteins (Additional file 3: Table S2). This list includes all the proteins identified in the saliva of both sexes and was used as our reference library for the analysis of SWATH data.
Functional classification of these 259 proteins showed that the most numerous protein functional groups and families were the proteins involved in metabolic processes (n= 48), proteases (n= 28), antioxidants (n= 20), protease inhibitors (n= 17), and proteins with unknown function (n= 36) (Table 1). Typically, these families and groups are also the most abundantly represented in the sialomes of the soft and hard tick species analysed to date [15].
Results obtained by LC-MS/MS in DDA mode
Besides generating the spectral library, we have analysed individually each sample of female and male saliva by LC-MS/MS. For simplicity, the lists of protein identifications obtained in the analyses of the individual replicated samples were combined in two unique lists, one for each sex, which were later filtered by eliminating the redundancies and the non-annotated hits. Table 2 and Additional file 3: Table S2 show 195 and 64 proteins identified in male and female saliva, respectively. Of them, 36 proteins are shared by both sexes, and 159 and 28 are unique to males and females, respectively (Fig. 2).
The presence of some differences in protein composition between the saliva of O. moubata females and males can also be observed in the different protein band patterns shown by the saliva of each sex in SDS-PAGE (Additional file: Fig. S1), and it has already been described by Díaz-Martin et al. [21] who also confirmed the massive presence of lipocalin proteins in female saliva. The hyper-abundance of lipocalins would have hampered the detection of the less abundant proteins, which most likely would have remained below the detection limits of the assays. As proof of this was the fact that when the saliva samples were equalised and the “excess” of the hyperabundant proteins removed, the number of proteins identified in females increased very significantly [21, 28]. At that time, the work by Díaz-Martín et al. [21] provided interesting novel information on the saliva proteome of males and females and revealed for the first time for soft tick differences in the saliva composition between both sexes. However, this study had an inconvenient, the method applied to increase the number of identifications, namely, the protein equalisation with the ProteoMiner Kit (BioRad), precluded the quantification of its components.
In ixodid ticks, the observed differences between males and females in the saliva composition are not surprising since both the feeding behaviour and the anatomy and functions of the salivary glands of both sexes are different [29,30]. However, the differences reported between O. moubata males and females in the salivary composition were somewhat surprising since typically soft tick adults, and specifically O. moubata adults, do not show anatomical differences in their salivary glands and they feed similarly. They ingest equivalent amounts of blood relative to their body weight and do it for a similar time, about an hour. This means that both sexes are obliged to overcome the same barriers and host defensive responses to complete blood ingestion, so it would be expected that both sexes would use the same repertory of anti-defensive salivary proteins [21].
In the current study, the different set of proteins identified by LC-MS/MS in female and male saliva also suggest qualitative differences in the salivary composition between sexes, following that was reported [21]. However, it must be noted that the reference database used in the present study for protein identification, i.e. the O. moubata sialotranscriptome, was obtained from female salivary glands only. Consequently, it might be assumed (i) that most likely the majority of the proteins identified in the present study, including those found in males only, would also be present in female saliva, and (ii) that the differences observed between sexes may be due more probably to quantitative differences in expression than to real absence/presence of particular proteins in one or another sex. As we will see later, the results of SWATH-MS also lend support to this idea.
Accordingly, it can be assumed that part of the qualitative differences observed by Diaz-Martin et al. [21] between the proteomes of male and female saliva, in which only 5.2% of the identified proteins were common, would possibly be due to quantitative differences.
Comparing LC-MS/MS and SWATH-MS analyses
To increase the number of protein identifications in both female and male saliva and quantify the protein expression level we analysed the saliva samples by two methods: LC-MS/MS operated in DDA mode and the free-label quantitative method SWATH-MS that operates in DIA mode.
Mass spectrometry methods that operate in DDA mode are based on the random selection and fragmentation of a fixed number of peptide precursors, generally the most intense peptide ions. On the other hand, in a SWATH-MS capture, all ionised peptides of a given sample that fall within a specified mass range are fragmented in a systematic and unbiased fashion using rather large precursor isolation windows [23]. Several published SWATH studies have demonstrated that SWATH-MS increases the sensitivity and the reproducibility of protein and peptide identification across multiple replicates [31,32]. Therefore, SWATH-MS might identify and quantify a higher number of proteins expressed simultaneously in O. moubata male and female saliva than LC-MS/MS, which in turn allows for comparing protein expression levels between both sexes.
Accordingly, we first assessed the performance and reproducibility of both methods in the identification of the O. moubata salivary proteome by comparing the results obtained by LC-MS/MS and SWATH-MS for the three replicate samples of each sex.
Regarding performance, LC-MS/MS analysis of the female samples resulted in the identification of 64 salivary proteins, while SWATH-MS of these same samples identified up to 165 salivary proteins (Table 2, Additional file 3: Table S2). Up to 40 of these proteins were identified by both methods, 24 exclusively by LC-MS/MS and 125 exclusively by SWATH (Fig. 3A). In male saliva, 195 and 165 proteins were identified by LC-MS/MS and SWATH-MS respectively (Table 2, Additional file 3: Table S2). Up to 136 male proteins were identified by both methods, 59 solely by LC-MS/MS and 29 solely by SWATH-MS (Fig. 3A).
With a 62% increase in the number of proteins identified, our data suggest that in the context of the female saliva, SWATH-MS is superior to DDA mode. However, in the male saliva DDA identified 15% more proteins than SWATH-MS. These data indicate that the benefit of SWATH over DDA MS, in the number of identified proteins, is unique for saliva of females and supports the notion that the performance of these techniques may be dependent on the fluid or the tissue analysed, as has been recently observed [32].
To assess the reproducibility in protein identification of both methods, we compared the data from the three biological replicates of female (F1, F2, F3) and male (M1, M2, M3) saliva. Fig. 3B shows that the reproducibility of LC-MS/MS was 27.3% and 36.6% in female and male saliva, respectively. On the other hand, the SWATH-MS reproducibility reached almost 100% (97-99%) in both sexes (Fig. 3C). Therefore, SWATH-MS outperformed DDA in the reproducibility of proteins identified across all three technical replicate analyses, and these results are in good agreement with previous reports [31].
Additional file 3: Table S2 shows the global results from both MS methods, which jointly identified 299 salivary proteins using the sialotranscriptome of O. moubata females as a reference database [19].
Quantification of the proteins identified in the female and male saliva by SWATH-MS
As already noted, SWATH-MS is a type of DIA method of analysis used to evaluate quantitatively complex samples with high reproducibility [33].
Using this technique, we have identified and quantified 165 proteins in the saliva of both female and male ticks, which were later classified in 21 groups and families (Table 2, Additional file 4: Table S3). Not unexpectedly, these groups/families coincide with the groups/families more abundantly represented in the O. moubata sialotranscriptome [19]. The groups with the highest numbers of proteins were proteins involved in metabolic processes (n = 30) and protein modification (n = 12), proteases (n = 19), lipocalins (n = 11), antioxidants (n = 10), regulation (n = 10), and unknown function (n = 24).
Fig. 4 represents the expression levels of the protein groups/families in both sexes, calculated as the mean spectral signal peak area for female and male saliva (Additional file 4: Table S3). The 24 proteins with unknown function have been excluded from the pie charts, and the groups containing 5 or fewer proteins (proteins involved in metabolism, signal transduction, protein synthesis, extracellular matrix, proteasome machinery and transporters) have been merged in one group named “other”.
Female saliva is predominantly composed of lipocalins as this group of proteins constitutes 95% of the saliva protein mass (Fig. 4A). The group includes 11 lipocalins, being moubatin (Q04669) and the so-named salivary lipocalin TSGP1 (F6K8G8) the most abundant as they respectively account for 40.39% and 55.33% of the total protein mass of this group (Additional file 4: Table S3).
Lipocalins are a large multigene protein family having dual functions as histamine and serotonin scavengers and as modulators of vertebrate inflammation and immunity [15]. Moubatin belongs to a lipocalin clade that includes proteins that inhibit platelet and neutrophil aggregation by scavenging of thromboxane A2 (TXA2) and proteins that inhibit complement activation by sequestering the C5 component [34]. TSGP1 belongs to the serotonin and histamine-binding group of the soft tick lipocalins [35]. The current results confirm the previous report by Oleaga et al. [20] regarding the great abundance and numerous isoforms of TSGP1 discovered in the proteome of the salivary glands of O. moubata and support the notion that in O. moubata TSGP1 would be the main scavenger of histamine and serotonin. These pro-inflammatory biogenic amines accumulate at the tick-feeding site and need to be efficiently removed for the tick to successfully feed [33]. Regardless of its function, TSGP1 is highly immunogenic and strongly recognised by the serum from pigs bitten by O. moubata, which has made it a useful tool for serological diagnosis of parasitism by O. moubata [36]. In fact, a recombinant form of TSGP1 has been already used in several seroepidemiological studies of pig exposure to this tick in Madagascar, Mozambique and Nigeria [37-39].
The remaining non-lipocalin proteins represent only 5% of the total protein content of female saliva. Among them, proteases, protease inhibitors, antioxidants, and proteins involved in the metabolism of carbohydrates and lipids are the following more abundant groups representing between 0.82 and 0.42% of saliva protein content (Fig. 4A). In these groups, the more abundant proteins were a carboxipeptidase (B7QF76), a metalloprotease (Q09JT3), SCO-spondin-like (XP_021004313), enolase (D4P967), aldehyde dehydrogenase (B7QAL5), catalase (A0A2U8T6B2) and phospholipase A2 (M9W8K4) (Additional file 4: Table S3).
For several of these proteins, classified as housekeeping proteins, it is well established that they can also play important extracellular functions at the host-parasite interface, helping ticks to feed [40, 41]. For instance, in O. moubata, salivary enolase acts as a pro-fibrinolytic plasminogen activator receptor [40] and salivary secreted phospholipase A2 plays as an antagonist ligand of host P-selectin preventing P-selectin mediated endothelial activation [43].
In quantitative terms, the proteome of male saliva is remarkably different from that of female saliva (Fig. 4B). In male saliva, lipocalins are also the most abundant proteins -though they only account for 27.12% of the saliva protein content- and TSGP1 (F6K8G8) is also the most abundant individual lipocalin accounting the 60% of the protein content of this group (Additional file 4: Table S3). The following more abundant groups were protease inhibitors (17.55%) and the proteins involved in carbohydrate metabolism (15.71%) (Fig. 3B), with serpin-2 (Q06B74) (52.27%) and enolase (D4P967) (55.72%) being their most abundant individual components, respectively.
Serpin 2 is a well-characterised serine protease inhibitor that inhibits trypsin and thrombin, and interferes with platelet aggregation and blood clotting [44,45]. This activity and the above-referred pro-fibrinolytic activity of enolase [42] would collaborate in maintaining host blood fluidity helping tick to feed.
Among the 165 proteins quantified by SWATH-MS in the saliva from males and females, 53 were differentially expressed (p < 0.05) between the sexes (Table 3); 13 proteins were over-expressed in females and 40 were overexpressed in males. The signal peak areas of the differentially expressed proteins in each of the samples analysed were shown using a heat map after z-score normalisation, using Euclidean distances. The hit map shows two main clusters comprising the F1–F3 samples and M1–M3 samples, which correspond to the saliva of females and males respectively (Additional file 5: Fig. S2).
Fig. 5 represents the top 10 proteins that are differentially (p < 0.05) overexpressed in the saliva of female or male ticks. As expected according to the above-reported results, the top 10 overexpressed proteins in females were 5 lipocalins, including moubatin and TSGP1, phospholipase A2, apyrase, a metalloprotease, a salivary secreted basic tail protein and a salivary basic tailless protein (Table 3). Apyrase has been identified in the saliva of most hematophagous vectors including soft and hard ticks. It is an enzyme that hydrolyses ATP and ADP to AMP and prevents platelet and neutrophil aggregation and thrombus formation, facilitating blood feeding [46,47]. A recombinant form of the salivary apyrase of O. moubata induced protective, strong humoral responses in animal vaccine trials that reduced tick feeding and survival [46].
In O. moubata, as in other ixodid and argasid tick species, metalloproteases are one of the enzyme classes most abundantly represented in the saliva [14,19,48, 49]. In this study, LC-MS/MS and SWATH-MS identified 30 proteases, 16 of them metalloproteases and 5 differentially expressed (p < 0.05) between females and males (Table 3).
Concerning basic tail and tailless proteins, their finding among the overexpressed proteins is not unexpected, as they are protein families abundantly found in the sialotranscriptomes of ixodid and argasid ticks, which suggest that they would play important and specific roles at the tick-host feeding interface [14,19,48, 49].
On the other hand, the top 10 proteins overexpressed in male saliva were two superoxide dismutases, two metabolic enzymes (adenosine amidase, hydroxypyruvate reductase), two proteins involved in immune mechanisms (gamma-interferon inducible lysosomal thiol reductase, spätzle alternatively spliced isoform), three proteins with unknown function, and ixodidin (Table 3 and Fig. 5). This last protein is an inhibitor of serine proteinases that shows antimicrobial activity [50].
As a whole, these results show that at least 165 out of 299 of the salivary proteins identified in the current study are shared by both sexes, which significantly reduces the range of qualitative differences between male and female saliva observed in previous works, where only 5.2% of the identified proteins were found in both sexes [21]. However, these results keep showing remarkable differences in the ratios of salivary proteins that males and females secrete in their saliva, which raises the question of the biological significance of these differences. It could be speculated that it may be related to the post-feeding blood processing or attraction and mating [51], but neither our current results nor a revision of the literature offered evidence to support or rule out such a notion and shed light on this matter.