DOI: 10.25345/c5t96b
title: MassIVE MSV000084579 - Structure and function of brain derived Orb2 amyloid filaments linked to memory
title: To isolate the different brain-derived Orb2 species, we used approximately 3 million 3 to 7- day-old adult Drosophila melanogaster Canton-S flies. First, flies were collected in 50 ml tubes and snap-frozen in liquid nitrogen. The heads were separated from the body by vortexing for 5-10 sec, and immediately snap-frozen in liquid nitrogen. The vortex and freezing cycles were repeated 3 times. The heads were collected in 50 ml tubes and stored at -80 C until their processing. Every 50 ml of fly heads was homogenized in 100 ml of lysis buffer (PBS, pH7.21, 5 percent glycerol, 0.1 Triton X-100, 1 percent NP40, 150 mM NaCl, 1 mM magnesium acetate, 1 mM dithiothreitol DTT, 1 mM PMSF and EDTA-free protease inhibitor (Roche)) with a Polytron Homogenizer and the homogenized tissue was rotated for 45 min at 4C. The homogenate was transferred to JA-25.50 tubes and centrifuged at 75,000g at 4C for 30 min. The obtained supernatant was stored for further processing while the obtained pellet was dissolved in additional 50 ml of lysis buffer and processed again with the Polytron Homogenizer following the same protocol. This procedure was repeated 3 times to ensure the maximum Orb2 protein recovery. The total volume of homogenate was then filtered through a Miracloth (Millipore) membrane to remove lipids, sonicated ten times for ten seconds and passed through a 0.45 micrometer filter. Using Ni-affinity, to exploit the presence of four consecutive histidines at the N-terminal region of Orb2, and other chromatography techniques, we purified from total head extracts. Approximately 10 micrograms of total protein, in liquid form, was precipitated for ID mass spectrometry analysis using a 4:1 volume MeOH/CHCl3 extraction procedure using Multidimensional Protein Identification technology (MudPIT). Multidimensional Protein Identification Technology- MeOH/CHCl3 -precipitated protein pellets were solubilized using Tris-HCl pH 8.5 and 8 M urea, followed by addition of TCEP (Tris(2-carboxyethyl) phosphine hydrochloride; Pierce) and CAM (chloroacetamide; Sigma) were added to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C at 1:100 w/w (Roche) at 37oC overnight. The samples were brought to a final concentration of 2 M urea and 2 mM CaCl2 and a second digestion was performed overnight at 37oC using trypsin (Roche) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The digested size exclusion eluates were loaded on a split-triple-phase fused-silica micro-capillary column and placed in-line with a linear ion trap mass spectrometer (LTQ, Thermo Scientific), coupled with a Quaternary Agilent 1100 Series HPLC system. A fully automated 10-step chromatography run was carried out. Each full MS scan (400-1600 m/z) was followed by five data-dependent MS/MS scans. The number of the micro scans was set to 1 both for MS and MS/MS. The settings were as follows: repeat count 2; repeat duration 30 s; exclusion list size 500 and exclusion duration 120 s, while the minimum signal threshold was set to 100. MS Data Processing- The MS/MS data set was searched using ProLuCID against a database consisting of 21,402 non-redundant Drosophila melanogaster proteins (downloaded from NCI RefSeq 2013-02-20), 177 usual contaminants, and, to estimate false discovery rates (FDRs), 21,579 randomized amino acid sequences derived from each NR protein entry. To account for alkylation by CAM, 57 Da were added statically to the cysteine residues. To account for the oxidation of methionine to methionine sulfoxide, 16 Da were added as a differential modification to the methionine residue. Peptide/spectrum matches were sorted and selected and compared using DTASelect/Contrast