MassIVE MSV000084579

Complete Public PXD016266

Structure and function of brain derived Orb2 amyloid filaments linked to memory

Description

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 [doi:10.25345/C5T96B] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: amyloid ; cryo-EM ; prion-like protein ; memory ; Orb2 ; CPEB

Contact

Principal Investigators:
(in alphabetical order)
Anita Saraf, The Stowers Institute for Medical Research, United States
Submitting User: asaraf
Number of Files:
Total Size:
Spectra:
Subscribers:
 
Owner Reanalyses
Experimental Design
    Conditions:
    Biological Replicates:
    Technical Replicates:
 
Identification Results
    Proteins (Human, Remapped):
    Proteins (Reported):
    Peptides:
    Variant Peptides:
    PSMs:
 
Quantification Results
    Differential Proteins:
    Quantified Proteins:
 
Browse Dataset Files Browse Results
 
FTP Download Link (click to copy):

- Dataset Reanalyses


+ Dataset History


Click here to queue conversion of this dataset's submitted spectrum files to open formats (e.g. mzML). This process may take some time.

When complete, the converted files will be available in the "ccms_peak" subdirectory of the dataset's FTP space (accessible via the "FTP Download" link to the right).
Number of distinct conditions across all analyses (original submission and reanalyses) associated with this dataset.

Distinct condition labels are counted across all files submitted in the "Metadata" category having a "Condition" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct biological replicates across all analyses (original submission and reanalyses) associated with this dataset.

Distinct replicate labels are counted across all files submitted in the "Metadata" category having a "BioReplicate" or "Replicate" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct technical replicates across all analyses (original submission and reanalyses) associated with this dataset.

The technical replicate count is defined as the maximum number of times any one distinct combination of condition and biological replicate was analyzed across all files submitted in the "Metadata" category. In the case of fractionated experiments, only the first fraction is considered.

"N/A" means no results of this type were submitted.
Originally identified proteins that were automatically remapped by MassIVE to proteins in the SwissProt human reference database.

"N/A" means no results of this type were submitted.
Number of distinct protein accessions reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct unmodified peptide sequences reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct peptide sequences (including modified variants or peptidoforms) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Total number of peptide-spectrum matches (i.e. spectrum identifications) reported across all analyses (original submission and reanalyses) associated with this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins quantified across all analyses (original submission and reanalyses) associated with this dataset.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
Number of distinct proteins found to be differentially abundant in at least one comparison across all analyses (original submission and reanalyses) associated with this dataset.

A protein is differentially abundant if its change in abundance across conditions is found to be statistically significant with an adjusted p-value <= 0.05 and lists no issues associated with statistical tests for differential abundance.

Distinct protein accessions are counted across all files submitted in the "Statistical Analysis of Quantified Analytes" category having a "Protein" column in this dataset.

"N/A" means no results of this type were submitted.
This dataset may not contain all raw spectra data as originally deposited in PRIDE. It has been imported to MassIVE for reanalysis purposes, so its spectra data here may consist solely of processed peak lists suitable for reanalysis with most software.