Journal of Proteome Research ARTICLE we measured the levels of cholesterol in the midgut extracts, reported as nanograms of cholesterol per microgram of protein. the supernatant was collected, dried, and resuspended in 2% acetonitrile, 0.1% TFA for LC MS/MS analysis. Brush Border Microvilli Vesicles (BBMV) Preparations Approximately 300 400 female mosquito midguts were transferred to 200 μL of microvilli buﬀer (50 mM D-mannitol, 20 mM Tris-HCl, pH 7.4, protease inhibitor cocktail (Sigma), 1 mM PMSF, 3 mM imidazole-HCl (Sigma). The midguts were then homogenized on ice using 30 strokes of a Dounce homo- genizer. The sample was then brought up to 10 mL in microvilli buﬀer and 0.05 g of MgCl2 were added to sample and mixed by vortexing. After a 20 min incubation on ice, the sample was centrifuged at 805 g for 10 min at 4 C. The supernatant was saved and the pellet was resuspended again in 10 mL of microvilli buﬀer and extracted two more times as described above. The supernatants from all extractions were transferred to a high speed centrifuge tube and subjected to ultracentrifugation at 30 000 g for 1 h at 4 C. The resulting supernatant was discarded and the pellet was resuspended in a ﬁnal volume of PBS equivalent to 1 μL PBS/midgut. Protein quantiﬁcation of all protein samples for each biological replicate was performed using a BCA Protein Assay Kit (Pierce, Rockford IL) according to the manufacturer’s instructions. Preparation of GPI-Anchored Protein Enriched Fractions The three biological replicate BBMV samples prepared from An. gambiae midguts were treated with 1 Unit of Bacillus cereus glycosylphosphotidyl inositol-speciﬁc phospholipase C (PI-PLC) (Sigma) at room temperature overnight. The sample was then centrifuged at 17 000 g for 10 min and the supernatant contain- ing glycosylphosphatidylinositol (GPI) anchored proteins was collected. Trypsin Digestion For analysis of the DRM fractions, 10 μg of protein from each fraction (fraction 4, 5, and 6) was dried by vacuum centrifugation and the sample was then resuspended in 20 μL of 20 mM NH4HCO3 (Sigma). We added 1 μL of 50 mM dithiothreitol (DTT) (Invitrogen) to the sample prior to incubation for 15 min at 60 C. We then added 5 μL of 22 mM iodoacetamide (Sigma) to the sample followed by incubation for 25 min at room temperature in the dark. Proteomics grade trypsin (Sigma, cat. # T6567) was added to sample (1:50) and incubated at 37 C for overnight. For GPI-anchored proteins samples, an in-gel diges- tion protocol was used. Approximately 10 μg of an enriched GPI- anchored protein sample was run on a NuPAGE 4 12% gel (Invitrogen), using 1 MES SDS NuPAGE running buﬀer (Invitrogen) for one hour at 200 V. Following overnight staining with Sypro Ruby (Invitrogen, Carlsbad, CA), and imaging on a 9210 Typhoon scanner (GE) (PMT of 500 V and 25 μM resolution), the entire lane was cut into 11 gel fragments. Gel pieces were dehydrated for 5 min in 200 μL of acetonitrile: 50 mM NH4HCO3. Gel pieces were dried in a vacuum centrifuge and then rehydrated in 100 μL of 25 mM DTT in 50 mM NH4HCO3 and incubated for 20 min at 56 C. The supernatant was discarded and 100 μL of 55 mM iodoacetamide in 50 mM NH4HCO3 was added to gel pieces and incubated at room temperature in the dark. The supernatant was discarded and gel pieces were washed in 1 mL of Milli-Q water. Next, the super- natant was discarded and gel fragments were dehydrated as described above. Gel pieces were rehydrated in 75 μL of 0.01% ProteaseMax containing 2 ng/μL trypsin. Gel pieces were incubated at 37 C for 3 h. Following a brief centrifugation, Peptide Fractionation For each DRM preparation, the digested, dried peptides were separated using an Agilent 3100 OFFgel fractionator (Agilent, Santa Clara, CA). Samples were loaded onto a rehydrated 24 cm pH 3 10 IEF strip (GE Healthcare, Piscataway, NJ) in 0.5% ampholytes in water. The peptides were separated for 50 kVhrs at 20 C, with maximum values of 4500 V, 50 μA and 200 mW. Paper wicks were exchanged as per standard protocols. Upon focusing, samples were removed from the sample wells, acidiﬁed with formic acid, and dried using a vacuum centrifuge. Peptides were resuspended in 8 μL 2% acetonitrile, 0.1% (w/v) triﬂuor- oacetic acid for LC MS/MS analysis. LC MS/MS Biological replicates were analyzed independently as follows. The entire 8 μL sample was injected onto an Agilent LC MS system comprised of a 1200 LC system coupled to a 6520 Q-TOF via an HPLC Chip Cube interface. The sample was trapped and analyzed using an Agilent large capacity HPLC chip (160 nL, 300 Å C18 trap with a 150 mm, 300 Å C18 analytical column). Peptides were loaded onto the chip using 97% solvent A (0.1% formic acid in water) and 3% solvent B (0.1% formic acid in 90% acetonitrile) at a ﬂow rate of 3 μL/min. Elution of peptides from the analytical column was done using a stepped gradient starting at 97% A at 300 nL/min. A linear gradient from 10 to 40% solvent B was applied from 2 to 80 min. The gradient was then stepped to 99% B and back to 97% A at 90 min. A 5-min re-equilibration step was applied to the end of each run. Column ﬂow throughout the run was analyzed by the 6520 Q-TOF in 2 GHz data dependent (autoMS2) mode with internal reference masses (m/z 391.28 and 1222.99). Precursor MS spectra were acquired from m/z 300 to 1600 and the top 4 peaks (+2, +3 and > +3 charge states, intensity >1000) were selected for MS/MS analysis. Product scans were acquired from m/z 50 to 1600 at a scan rate of 1.7/second. A medium isolation width (∼4 amu) was used, and a collision energy of slope 3.9 V/100 Da with a 2.9 V oﬀset was applied for fragmentation. A dynamic exclusion list was applied, with precursors excluded of 0.13 min after one MS/MS spectrum was acquired. Data Analysis The data acquired from Agilent Mass Hunter 2.3.0 was searched against the Anopheles gambiae proteome fasta database (An. gambiae P6 release, VB-2011 04) downloaded from Vec- torBase (http://www.vectorbase.org/GetData/Downloads/). The data was ﬁrst searched with multiple search engines (Mascot,17 OMSSA18 and X!Tandem19 with native scoring) with the following parameters: carbamidomethylation and oxi- dized methionine set as variable modiﬁcation, mass tolerances on precursor and fragment ions set as 30 and 20 ppm respectively and missed cleavage as 1. We then combined these search results by a meta-search methodology. MS raw ﬁles were converted to mzXML format using Trapper (ISB) and searched by PepArML which uses an unsupervised, model-free, combining framework.20 All of the computed search results were combined by the PepArML result combiner, employing a random forest machine learning technique, based on the Weka software package,21 to generate a consensus set of peptide-spectrum matches. Multiple decoy searches were also used to estimate the statistical signiﬁcance of the ﬁnal resulting protein identiﬁcations. Two decoy database 5152 dx.doi.org/10.1021/pr2006268 |J. Proteome Res. 2011, 10, 5150–5162