Journal of Proteome Research cell types.7,8 Components of the cytoskeleton have also been demonstrated to associate with DRMs in situ36 and that the presence of these proteins is not surprising given the dynamic interactions occurring between lipid rafts and the cytoskeleton and cytoskeleton associated proteins. Previous analyses of an enrichment of midgut brush border microvilli (BBMV) mem- branes, which encompasses the same apical membrane face represented by our DRM fractions, also identiﬁed cytoplasmic proteins and cytoskeletal proteins.37 39 At present it remains unclear which speciﬁc cytosolic proteins are mechanistically associated with DRMs and which are simply detergent-resistant contaminants. However, we noted that common DRM-asso- ciated proteins such as stomatin,39 galectin-4 like proteins,40 annexin-2,41 alkaline phosphatase,42 and protein 14-3-343 were all detected in our midgut DRM fractions. Also, proteins such as AgMuc1 (AGAP001192) and AgDuox (AGAP009978) were not detected in the DRM fractions illustrating that as expected not all midgut surface proteins are present in the DRM fractions. To estimate the number of midgut DRM proteins that are potentially surface-associated, the amino acid sequences of all 1452 proteins were analyzed for the presence of a secretory signal sequence by SignalP (Table S1, Supporting Information). Many surface-associated proteins contain a signal sequence, which allows proteins to enter the secretory pathway and be traﬃcked to the plasma membrane. Of the 1452 proteins, 280 had a putative N-terminal signal sequence (Table S1, Supporting Information). However, not all surface-associated proteins con- tain a canonical signal sequence. Galectins, annexins, and sto- matin were detected in our DRM fractions and categorized as extracellular proteins despite the lack of a signal sequence since these proteins are frequently surface-associated proteins in other organisms.44,45 Furthermore, not all proteins with a signal sequence are surface-associated; thus, based on sequence homol- ogy and evidence in the literature, 89 intracellular proteins, which had a predicted signal sequence, were excluded from the extra- cellular DRM protein list. Altogether, a total of 191 DRM- associated proteins (FDR < 1%) were predicted to be extra- cellular and exposed to the midgut lumen (Table S2, Supporting Information). These 191 proteins were further classiﬁed accord- ing to predicted functional domains (Figure 2b). A heat map representing the spectral assignment for each extracellular pro- tein for every DRM preparation is provided; demonstrating reproducible protein identiﬁcations across independent prep- arations for a majority of the proteins (Figure S3, Supporting Information). These ﬁndings are particularly relevant to our eﬀort to identify midgut surface molecules as potential TBV candidate antigens. Mosquito TBVs based on midgut surface proteins, rely on the ability of antibodies to bind their target on the lumenal surface of the midgut epithelium subsequently following ingestion by a mosquito that took its bloodmeal from an immunized vertebrate host. To date, only six proteins have been studied as candidate TBV antigens (Table 1), by virtue of their knockdown by RNAi or direct targeting by antibodies. A heat map, highlighting the currently recognized TBV candidate antigens, as well as a subset of canonical lipid raft protein markers and potential TBV targets are shown in Figure 2c. From this analysis alone, the number of novel, putative vaccine targets has increased 27-fold. Of the 191 predicted extracellular DRM-associated proteins, the common classes of proteins included: peptidases (27.7%), glycosyl hydrolases (8.4%), protease inhibitors (1.6%), lectins (8.9%), proteins involved in cell adhesion (6.3%), receptors (3.7%), proteins of unknown function (13.6%), transporters ARTICLE (1.6%), proteins containing an immunoglobulin-like domain (2.6%) and proteins classiﬁed as “other” which did not fall into any other category (25.1%) (Figure 2b and Table S3, Supporting Information). Previous studies have reported that DRM fractions from BBMVs are enriched in brush border enzymes.42 These results are consistent with our ﬁndings, in which peptidases, both soluble and membrane bound, comprised 27.7% of the total predicted extracellular midgut DRM-associated proteins. The most common peptidases were trypsin-like and M1 membrane alanyl aminopeptidases such as AgAPN1 (Table S2, Supporting Information). A second group of enzymes detected in the DRM fractions are glycosyl hydrolases. These enzymes, which catalyze the release of glycans via hydrolysis of glycosidic bonds, have been reported to be present on the surface of cells46 and adult mosquito midgut BBMVs,11 as well as secreted into the lumen of the mosquito midgut during blood digestion.47 Also, protease inhibitors, including Serpin-4 (AGAP009670), were detected in the DRM fractions. Serpins belong to a large family of serine protease inhibitors that regulate mosquito innate immune functions.48 Lectins, a class of proteins that bind to speciﬁc glycans, were also detected in the DRM fractions and included ﬁve galectins and seven peritrophins. Galectins are a family of lectins that have aﬃnity for β-galactosides, and some members of this family, such as galectin-4, have been shown to stabilize lipid rafts by forming a lattice-like network of galectin-glycan interactions.40 In addition, peritrophins, which are proteins with chitin-binding domains and are associated with the peritrophic matrix (PM),49 were detected in our DRM fractions despite the fact that our preparations used sugar-fed midguts that lack a peritrophic matrix. However, this observation is consistent with the report that PM proteins are synthesized and stored in secretory vesicles just beneath the apical plasma membrane before ingestion of a blood meal; thus, their proteomic detection in sugar-fed midguts is expected.50 Moreover, DRMs and DRM- associated proteins have been shown to mediate secretory vesicle exocytosis and are hypothesized to cooperatively tether secretory vesicles and granules to “active zones” in the plasma membrane for rapid release.51 Proteins categorized as adhesins included cadherin-like proteins, integrins, CD36 scavenger receptor homologues, and proteins containing a laminin domain. Integ- rins belong to a family of proteins that function to connect components of the extracellular matrix/glycocalyx to the cell cytoskeleton and play roles in cell cell adhesion and signal transduction.52 While many integrins are associated with the basal lamina, there are reports of integrins being associated with DRMs on the apical membrane.53 Proteins categorized as receptors included two mannose-6-phophate receptors, an in- sulin-like growth factor binding protein, and a low-density lipoprotein receptor. Lastly, among the proteins of unknown function, AGAP000570 was previously reported as the most abundant protein in the An. gambiae PM proteome.49 However, it is hypothesized that midgut microvilli are closely intercalated with the developing PM ﬁbrils, which may implicate AGAP000570 as a tethering anchor for the PM. Thus, its inclusion in the PM proteome may be artiﬁcial, resulting from the dissection of the PM from the midgut epithelium during sample preparation.49 Immunoglobulin-like Proteins A total of 29 proteins containing at least one immunoglobulin- like domain were detected in the DRM fractions (Table 2). Of the 29 immunoglobulin domain-containing proteins, 20.7% contained a signal sequence and therefore are potential secreted 5156 dx.doi.org/10.1021/pr2006268 |J. Proteome Res. 2011, 10, 5150–5162