Experimental Procedure
Materials
Reagents for 2-DE were purchased mainly from Amersharm Pharmacia Biotechnology (Uppsala, Sweden). Gel-CODE glycoprotein-staining kit was from Pierce (Rockford, IL, USA). Other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) unless specified.
Sample Preparation
The wild type strain (Canton S) of Drosophila melanogaster, raised on normal cornmeal-agar media, was used in this study. As previously described [Matsumoto et al., 1982; Takemori et al., 2006], adult flies were frozen in liquid nitrogen, immediately immersed into cold acetone, and dehydrated at -20 oC for 1 week. Forty brains and 150 compound eyes were dissected under a microscope. For 2-DE, dissected tissues were homogenized with 100 l of a lysis solution containing 8.5 M urea, 4% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 0.2% (w/v) Bio-Lyte 3/10 (BIO-RAD, Hercules, CA, USA), and 5% (v/v) -mercaptoethanol. After centrifugation at 15,000 x g for 7 min, the supernatants were loaded on to an isoelectric focusing gel.
Two-Dimensional Gel Electrophoresis
Isoelectric focusing gel electrophoresis was carried out using Pharmacia IPGphor with a cup loading strip holder. Immobiline Dry Strips (pH 3-10, 13 cm in length) were rehydrated for 15 hr at room temperature with 250 l of lysis solution. Electrophoresis was performed at 500 V for 5 min, 4,000 V for 1.5 hr, and 8,000 V up to a total of 20,000 Vhr. The strips with proteins at their isoelectric focusing points were incubated for 30 min in sodium dodecyl sulfate (SDS) equilibration buffer [50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 2% (w/v) dithiothreitol]. The isoelectric focusing strips were then embedded in a 4% acrylamide stacking gel casted over an 11% SDS-polyacrylamide gel (135 mm x 195 mm x 1.0 mm). Following electrophoresis, gels were stained either with Coomassie blue for visualization of protein spots.
In-Gel Tryptic Digestion
Protein spots were excised from gels and subjected to in-gel digestion with sequence-grade modified trypsin (Catalog # V5111; Promega, Madison, WI) as previously described in detail [Matsumoto and Komori, 2000; Takemori et al., 2006] with minor modification. The tryptic digests were reconstituted with 0.2% (v/v) trifluoroacetic acid for mass spectrometric analysis.
Mass Spectrometry
Peptide mass fingerprinting (PMF) analysis was performed by MALDI-TOF mass spectrometer (Voyager Elite; PerSeptive Biosystems, Framingham, USA). Monoisotopic peaks of detected peptide ions were assigned by PerSeptive GRAMS/386 version 3.02.
Protein Identification
PMF data were submitted to MASCOT peptide mass fingerprint program (Matrix Science, London, UK) in order to obtain a protein candidate for each protein spot. Database searches were performed against the National Center for Biotechnology Information (NCBI) nonredundant database version 20061027 using the following parameters; (1) the protein database under Drosophila (51,498 sequences), (2) unlimited protein molecular weight and pI ranges, (3) presence of protein modifications including acrylamide modification of cystein, methionine oxidation, protein N-terminus acetylation, and pyroglutamic acid, and (4) peptide mass tolerance of ~ 0.25 or 0.5 Da. After matching experimental peptide mass values against predicted peptide masses of each entry in the database, MASCOT calculates the probability-based MOWSE score that is a measure of the statistical significance of the first protein candidate, and scores > 60 represent p < 0.05 in our case. For the identified proteins, we have collected the bioinformatics of their biological/physiological functions using FlyBase genomic database (http://flybase.bio.indiana.edu).
Reference
Matsumoto, H., OfTousa, J. E., and Pak, W. L. (1982) Light-induced modification of Drosophila retinal polypeptides in vivo. Science 217, 839-841.
Matsumoto H, Komori N. Ocular proteomics: cataloging photoreceptor proteins by two-dimensional gel electrophoresis and mass spectrometry. Methods Enzymol. 2000;316:492-511.
Takemori N, Komori N, Matsumoto H. Highly sensitive multistage mass spectrometry enables small-scale analysis of protein glycosylation from two-dimensional polyacrylamide gels. Electrophoresis. 2006 Apr;27(7):1394-406.
