Journal of Proteome Research ARTICLE Figure 1. Isolation and puriﬁcation of Anopheles gambiae adult midgut Detergent Resistant Membranes (DRMs). (a) Experimental work-ﬂowchart for the isolation of DRM fractions of An. gambiae midguts. For each biological replicate, approximately 1000 mosquitoes midguts were dissected then homogenized in 4 C with 1% Triton X-100. Samples were layered on a sucrose gradient, centrifuged at 100 000 g for 18 h. Fractions were TCA/ acetone precipitated, digested with trypsin, and analyzed by LC MS/MS. (b) Photograph of the DRM layer in sucrose gradient following ultracentrifugation. Regions IV, V, VI correspond to fractions 4, 5, and 6. (c) SDS-PAGE SYPRO Ruby stained gel. Lane (1), molecular weight markers (kDa), Lane (2), 5 μg/well of fractions 4, 5, and 6 (DRM fractions). (d) Immunoblot analysis of An. gambiae adult midgut DRMs shows an enrichment of AgAPN1 in DRM Fractions. Following sucrose density gradient ultracentrifugation, 11 1-mL fractions and a pellet were collected. Equal amounts (20 μg) were probed for AgAPN1 and AgEcad with the respective antibodies. Fractions 4/5/6 (bold) represent the pooled DRM fraction (see text for details). Near infrared ﬂuorescence quantiﬁcation expressed as integrated intensity as measured by the Odyssey System (LI-COR) for each of the three respective AgAPN1 protein bands (Bands A, B and C) present across diﬀerent fractions is indicated in boxed rows. The quantiﬁcation data suggest AgAPN1 is more concentrated in fraction 4/5/6 (lane 1) and pellet as compared to other fractions (fractions 7 and 8; fractions 9 and 10; and fraction 11). other dietary sterol precursors.32,33 Additionally, a sterol carrier protein-2 homologue was found in A. aegypti and shown to facilitate cholesterol uptake in cultured cells.34,35 To our knowledge, there is no published report conﬁrming the presence of choles- terol in female adult An. gambiae midguts. Since cholesterol is a key component of DRMs, we analyzed fractions from the sucrose gradient to determine if cholesterol is present and enriched in the DRM fractions (fractions 4, 5, and 6). Samples of the midgut Triton X-100 lysate before ultracentrifugation were used to determine the total amount of cholesterol. We estimated the presence of approximately 12.17 ng cholesterol/μg of protein in our total midgut lysate from two biological replicate samples. Next, to conﬁrm enrichment of cholesterol in the putative DRM fraction, samples from fractions 4, 5, and 6 postultracentrifugation were analyzed. These putative DRM fractions had an averaged 4.41 fold increase in cholesterol/μg protein as compared to the total midgut lysate as determined from two biological replicate samples. These data complement our visualization of DRMs after ultra- centrifugation (Figure 1b) and suggest that fractions 4, 5, and 6 are enriched in DRMs. It is important to note that the assay used cannot distinguish between cholesterol and cholesterol esters. Proteomic Analysis and Classification of An. gambiae Mid- gut Detergent Resistant Membrane-Associated Proteins To capture the midgut DRM proteome we used a complex sample fractionation strategy coupled with thorough mass spec- trometric analyses. After DRM enrichment, replicate samples were digested using standard protocols and then heavily sepa- rated by peptide-level isoelectric focusing into 24 fractions followed by LC MS/MS. This approach was used to increase the probability of achieving greater coverage of proteins and allowed us to detect more low-abundant proteins. The advantage of such an approach is that with a greater coverage, a more detailed atlas of the DRM proteome can be generated. We used a combination of multiple search engines and machine learning recombination to maximize the number of assigned spectra and minimize the false discovery rates. Further- more, to minimize any protein redundancy in our protein assignments, an advanced clustering method was used (see Methods). Using this advance informatics workﬂow, we identi- ﬁed 1452 proteins using a highly stringent cutoﬀ of <1% FDR (false discovery rate) (Table S1, Supporting Information). Given the number of spectra assigned, we were able to use agglom- erative hierarchical clustering (AHC) analysis to determine the degree of variability between the three biological DRM preps (Table S2, Supporting Information). This method takes into consideration distance and relations of all of the factors, and is a much more robust method of determining reproducibility than Venn diagrams showing common proteins. This analysis showed a strik- ing degree of similarity (>80%) between the replicates that were the greatest distance from another. Given the potential degree of variability in fraction collection and preparation and the random nature of spectral acquisition in data-dependent MS/MS studies, this degree of similarity represented excellent reproducibility between biological replicates and permitted the pooling of the data sets. We manually curated all of the 1452 identiﬁed proteins to gain molecular insight for the proteins identiﬁed using our approach (Figure 2 and Table S1, Supporting Information). These 1452 5154 dx.doi.org/10.1021/pr2006268 |J. Proteome Res. 2011, 10, 5150–5162