Computational protocol: A Proteomic Investigation of Soluble Olfactory Proteins in Anopheles gambiae

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Protocol publication

[…] Along with our previously described protocol , the extracts were concentrated to 50 µL and then diluted to 250 µL with a buffer containing 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 1% (v/v) IPG buffer (GE-Healthcare) and 60 mM of Dithiothreitol (DTT). The samples were loaded by rehydration for 11.5 hours in IPG strips (pH 3–11, 7 cm). Isoelectrofocusing was performed with an Ettan IPG Phor III system (GE-Healthcare) using the following conditions: 50 V (2 hours), 100 V (2 hours), 500 V (2 hours), 1000 V (2 hours), 6000 V (1.5 hours). Strips were equilibrated for 15 minutes in a TrisHCl 1.5M pH 8.8 solution containing glycerol 29.3%, urea 6 M, SDS 2% (w/v), DTT 1% and then for further 15 minutes in a Tris-HCl 1.5M pH 8.8 solution, containing glycerol 29.3%, urea 6M, SDS 2% and Iodoacetamide 2.5%.Gels were stained using Brilliant Blue G-Colloidal Concentrate (Sigma). The excised spots were subjected to tryptic digestion and nano HPLC-ESI Orbitrap analyses. The acquired MS and MS/MS data were searched with Proteome Discoverer 1.2 (Thermo Fisher) using SEQUEST as the search algorithm against a database created by merging the sequences of the peptides predicted from An. gambiae genome (Anopheles_gambiae.AgamP3.48.pep.all.fa.gz, and Anopheles_gambiae.AgamP3.48.pep.abinitio.fa.gz downloaded at with the entries related to Anopheles in UniProtKB. Searches were performed allowing up to three missed cleavage sites, 10 ppm of tolerance for the monoisotopic precursor ion and 0.5 mass unit for monoisotopic fragment ions and carbamidomethylation of cysteine and oxidation of methionine as variable modifications. False discovery rate was set at 1%. [...] Samples for shotgun experiments were resuspended in 200 µL of urea containing buffer (8 M Urea, 100 mM TrisHCl, pH 8.5). Based on Bradford colorimetric assay, the samples of female and male antennal extracts contained 80 and 200 µg of total protein, respectively. Reduction of disulfide bridges and alkylation was performed by treating samples with 2 mM DTT (30 minute at 25°C), followed by 11 mM iodoacetamide (20 minutes at room temperature in the dark). LysC digestion was then performed by incubating the samples with LysC (Wako) in a ratio 1∶40 (w/w) under gentle shaking at 30°C. The digestion products were diluted 3 times with 50 mM ammonium bicarbonate and incubated with 10 µL of immobilized trypsin (Applied Biosystems) for 4 hours under rotation at 30°C.Fifteen 15 µg of each resulting peptide mixture were then desalted on Stage Tip and the eluates dried and reconstituted to 50 µL in 0.5% acetic acid. Fractions containing 7 µg of protein were injected.The extract was analysed on three sets of analyses, each performed in triplicates on a LC-MS/MS system (Eksigent nano Liquid Chromagrapher coupled to a Linear Trap Quadrupole -Orbitrap Velos (Thermo)), on a C18 (75 µm i.d.×15 cm, 1.8 µm, 100 Å) column at 250 nL/min using a 155 or 255 minutes gradient ranging from 5% to 60% of solvent B (solvent A = 5% acetonitrile, 0.1% formic acid; solvent B 80% acetonitrile, 0.1% formic acid). The nanospray source was operated with a spray voltage of 2.1 kV and ion transfer tube temperature of 275°C. Data were acquired in data dependent mode, with one survey MS scan in the Orbitrap mass analyzer (resolution 60,000 at m/z 400) followed by up to 20 MS/MS in the ion trap on the most intense ions (intensity threshold = 750 counts). Once selected for fragmentation, ions were excluded from further selection for 30 seconds, in order to increase new sequencing events. Raw data were analyzed using the MaxQuant proteomics pipeline (v1.2.2.5) and the ANDROMEDA search engine against the database described above. Carbamidomethylation of cysteines was chosen as fixed modification, oxidation of methionine and acetylation of N-terminus were chosen as variable modifications. The search engine peptide assignments were filtered at a False Discovery Rate <1% and the feature “match between runs” was not enabled; other parameters were left as default.For each set of analysis, relative abundance of proteins was estimated using the “Intensity” values as produced by MaxQuant software , normalised on the total intensity signal. The results of the three sets were averaged. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet, MaxQuant, Andromeda
Databases UniProtKB
Application MS-based untargeted proteomics
Organisms Anopheles gambiae