Journal of Proteome Research replicates were selected to increase the precision of the false discovery rate (FDR) estimation process. Proteins with at least 2 peptides at <1% FDR and peptide length >5 amino acids were stored for subsequent query and retrieval by Maspectras 2.22 Identiﬁcations that passed these criteria were then grouped accord- ing to shared peptides (MS-evidence grouping, to eliminate isoforms, splice variants and fragments which share peptides) and the representative protein of the group was considered as a unique protein identiﬁcation. The data analysis pipeline meets all MIAPE standards22 and has been uploaded to Tranche (https:// proteomecommons.org/tranche). The bioinformatics metho- dology was designed to obtain a maximum of true positives while minimizing false positives and false negatives. Several publications have detailed the need for several parallel search engine processes.23 25 As such, we used a meta-search strategy based on the PepArML result combiner. Our use of multiple search engines (Mascot, OMSSA, and X! Tandem) within the PepArML meta-search engine platform, substantially increased the number of proteins identiﬁed for each of the three representative DRM preparations (Figure S1, Supporting Information) to 1452 proteins at a stringent cutoﬀ of <1% FDR (Table S1, Supporting Information). As to the rate of peptides to spectra, PepArML framework identiﬁed 12 040 peptides for 167 045 spectra. For the GPI subproteome, the peptide assignments for GPI proteins are 393 peptides for 5392 spectra using PepArML. The manual curation of the 1452 identiﬁed DRM proteins was performed using the conserved domains as predicted by VectorBase (AgamP6, VB-2011 04), InterPro, Pfam, UniProt, and BLAST searching using the nonredundant protein sequences database on NCBI. If multiple diﬀerent conserved domains were listed in a single protein, the larger conserved domain was used to classify that protein. To predict which proteins are extracellular and exposed to the midgut lumen, only proteins which had a signal sequence (with the exception of galectins, annexins ANXB10B and ANXB9, and stomatin) and were not predicted to be intracellular based on sequence homology and annotation were counted. Agglomerative Hierarchical Clustering Agglomerative hierarchical clustering (AHC) was performed by normalizing the spectral counts for each protein observed obtained from Maspectras to the number of total spectra observed for that group, and expressed as a percentage, using Microsoft Excel for Windows 2007. AHC was calculated using XLstat specifying Euclidean distance by dissimilarity and using Ward’s method as the agglomeration method (Table S2, Sup- porting Information). Production of anti-AgEcad Antibodies The AgEcad primers for AGAP007203, (AgEcad Forward, 50-CAC CAT GGT TGG TAC GCC GGT ACT GCG CGT-30, AgEcad Reverse, 50-CTG CTC CAG CAG GAA TGT ATT TTT CGA-30) were used to clone a 779 base pair fragment of AgEcad into the TOPO vector pBAD202D (Invitrogen) and expressed in E. coli. AgEcad is a homologue of DE-cadherin/shotgun in Droso- phila, which is involved in adherens junction integrity between adjacent midgut epithelial cells and is distributed primarily along the basolateral domain of polarized cells.26,27 The resulting recom- binant his-tagged protein was puriﬁed using Ni-agarose beads (Qiagen). Rabbit polyclonal antiserum was generated against the puriﬁed recombinant protein (Washington Biotechnology, Inc.) and recognized a single protein band of the expected size. ARTICLE Immunoblot Analysis Approximately 20 μg of protein from each of the sucrose fractions were run in each lane of a 4 12% Bis-Tris Invitrogen gel (using conditions described above) and transferred to a nitro- cellulose membrane for one hour at 100 V at 4 C. Immunoblots were probed with either1:200 α-AgAPN1 (11) or 1:50 α-AgEcad. Primary antibodies were detected by α-rabbit secondary anti- bodies CW 800 (LI-COR) and detected on an Odyssey infrared imaging system. Quantiﬁcation of the APN1 bands was performed using the LI-COR Odyssey 3.0 application software. ’ RESULTS AND DISCUSSION To test our hypothesis, we isolated An. gambiae midgut DRM fractions from 1,000 An. gambiae sugar-fed midguts for each of our three biological replicate DRM preparations. In this study, we chose to use sugar-fed midguts as opposed to blood-fed midguts for the following reasons: (a) two previous studies have shown that several midgut surface proteins, including AgAPN1 (AGAP004809, Table 1), are expressed on the surface of sugar- fed midguts,11,28 and (b) by analyzing sugar-fed midguts, we avoided gross contamination of our midgut DRM sample with human blood and serum proteins, which would complicate the analysis. Very little is known about the protein expression proﬁle of the panoply of genes that have been shown by functional genomics analyses to be upregulated in response to ookinete invasion of the midgut.29,30 Therefore, given what is known about AgAPN1, we also sought to determine if these proteins are present in sugar-fed midguts. After ultracentrifugation of the Triton X-100 midgut lysate, we observed that lipid rafts are indeed visible at the interface between the 5 and 38% sucrose layers, represented as fractions 4, 5, and 6 (Figure 1a c and Figure S1, Supporting Information). Three independent DRM preparations were isolated for analysis by tandem mass spectrometry. GPI-anchored proteins, which are associated with DRMs are frequently used as markers of DRMs to conﬁrm DRM enrichment.4,5 To indirectly conﬁrm the presence of GPI-anchored proteins, immunoblots of fractions and the pellet from the sucrose gradient were probed with antibodies that recognized An. gambiae amino peptidase N, AgAPN1, a microvillar GPI-anchored glycoprotein, which is currently the leading mosquito based TBV target (Figure 1d).11 As expected, immunoblots probed with speciﬁc antisera to AgAPN1 recog- nized the predicted Mr = ∼125 kDa and protein double band at Mr = ∼60 kDa.11 These data indicate that AgAPN1 is present across all fractions, but is particularly concentrated in the DRMs (fractions 4, 5, and 6) and the pellet (Figure 1d). In addition, speciﬁc antibodies were raised against a recombinant fragment of the An. gambiae E-cadherin homologue (AgEcad, AGAP007203) of the adherens-junction DE-cadherin/shg in Drosophila.26,27 This protein was selected as a marker for non-DRM proteins and used to demonstrate the diﬀerential separation of DRM- associated proteins through sucrose gradient fractionation. Im- munoblots probed with AgEcad speciﬁc antisera revealed that AgEcad was detectable only in the pellet fraction (Figure 1e). Detection and Quantification of Cholesterol in An. gambiae Midguts Cholesterol, which is enriched in lipid rafts, is found in the membranes of almost all eukaryotes. Insects such as An. gambiae, do not synthesize cholesterol de novo as they lack the genes for key enzymes necessary for cholesterol biosynthesis.31 However, Aedes aegypti larvae are capable of synthesizing cholesterol from 5153 dx.doi.org/10.1021/pr2006268 |J. Proteome Res. 2011, 10, 5150–5162