MudPIT analyses of the proteins co-purified with wild type-Hzg-3XHA/Flag and phosphatase dead-Hzg-3XHA/Flag affinity purifications from Drosophila melanogaster S2 cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ
ORGANISM(S): Drosophila Melanogaster (ncbitaxon:7227)
SUBMITTER: 
Anita Saraf
PROVIDER: MSV000083305 | MassIVE | Tue Jan 08 14:54:00 GMT 2019
SECONDARY ACCESSION(S): PXD012273
REPOSITORIES: MassIVE
Publications
Amyloid-like Assembly Activates a Phosphatase in the Developing Drosophila Embryo.
Nil Zelha Z Hervás Rubén R Gerbich Therese T Leal Paulo P Yu Zulin Z Saraf Anita A Sardiu Mihaela M Lange Jeffrey J JJ Yi Kexi K Unruh Jay J Slaughter Brian B Si Kausik K
Cell 20190901 6
Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. Here, we report the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely be ...[more]
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Multidimensional Protein Identification Technology- TCA-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 (v. 1.3.3) 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 to an FDR less than 5% at the peptide and protein levels, using DTASelect in combination with swallow, an in-house software.
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Project description:Protein Complexes Purification- Drosophila S2 cells were stably-transfected with pRmHa3-based inducible expression vectors to produce epitope-tagged Non-stop-2xFLAG-2xHA (Non-stop-FH). Cells stably transfected with empty vector served as negative control. Protein complexes containing Non-stop were purified from nuclear extracts via their FLAG epitope tag. Isolated complexes were further separated by size using Superose 6 gel filtration chromatography. Eluates from the FLAG-IP and SEC purifications were TCA-precipitated from proteomics analysis. Three biological replicates of the SEC group 2 were analyzed.
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2018-07-17 | MSV000082625 | MassIVE
Project description:MudPIT analysis of Plasmodium polysomes. TCA precipitated pellet from Plasmodium falciparum polysomes (~50ug) was solubilized in TRIS-HCl pH8.5 and Urea; reduced and alkylated. The protein suspension was digested overnight using Endoproteinase Lys-C followed by a second overnight digestion at 37°C using Trypsin. Formic acid (5% final) was added to stop the reactions. Sample was loaded on split-triple-phase fused-silica micro-capillary column and placed in-line with linear Velos pro ion-trap mass spectrometer (Thermo Scientific), coupled with quaternary Agilent 1260 series HPLC. Fully automated 10-step chromatography run (for a total of 20 hours) was carried out on the sample. Each full MS scan (400-1600 m/z) was followed by ten data-dependent MS/MS scans. The number of the micro scans was set to 1 both for MS and MS/MS. The dynamic exclusion settings used were as follows: repeat count 2; repeat duration 30s; exclusion list size 500 and exclusion duration 90 sec, the minimum signal threshold was set to 500. The MS/MS dataset was searched using SEQUEST (v.27; rev. 9) against a database of 72358 sequences, consisting of 5487 P. falciparum non-redundant proteins (downloaded from PlasmoDB on 2012-07-12), 30536 H. sapiens non-redundant proteins (downloaded from NCBI on 2012-08-27), 177 usual contaminants (such as human keratins, IgGs, and proteolytic enzymes), and, to estimate false discovery rates, 36179 randomized amino acid sequences derived from each non-redundant protein entry. To account for alkylation by CAM, 57 Da were added statically to cysteine residues and to account for the oxidation of methionine residues to methionine sulfoxide (that can occur as an artifact during sample processing) 16Da were added as a differential modification to methionine residue. Peptide/spectrum matches were sorted, selected using DTASelect/CONTRAST (v 1.9). Proteins had to be detected by 1 peptide with 2 independent spectra, leading to FDRs at the protein and spectral levels of 2.89% and 0.26%, respectively.
2013-10-31 | PXD000553 | Pride
Project description:Yeast protein extractions and subcellular fractionation
Yeast cells expressing FLAG tagged proteins were grown in YPD medium at 30oC to OD600 of 0.8. 12 Liters YPD culture of cells were pelleted and washed in SB buffer. The pellet was resuspended in SB buffer and treated with 2 mg/ml Zymolyase 20T at 23oC for 45 min. The resulting spheroplasts were collected by centrifugation at 2,710 g for 5 min and washed twice with SB buffer. The spheroplasts were then resuspended in 20 ml of homogenization buffer (10 mM Tris-HCl pH 7.4, 0.6 M sorbitol, 1 mM EDTA, 0.2% (w/v) BSA) and homogenized with 8 strokes of a revolving pestle at full speed. The homogenized spheroplasts were centrifuged at 1,500 g for 5 min and the resulting pellets were collected for nuclear extracts while the supernatant was collected for mitochondrial extracts.
The pellet enriched for nucleus was washed with NP buffer (0.34 M sucrose, 20 mM Tris pH 7.5, 50 mM KCl, 5 mM MgCl2) and resuspended and homogenized in H350 buffer (40 mM HEPES-KOH pH7.5, 10% glycerol, 350 mM KCl, 2 mM MgCl2, 1 mM EDTA, 0.02% NP40, 1 ug/ml pepstatin A, 2 ug/ml leupeptin, 1 mM PMSF) with 25 strokes. The crude extract was incubated with 125 U of Benzonase on a roller for 1 hour in 4oC, then clarified by ultracentrifugation at 45,000 rpm for 90 min after following a centrifugation at 20,000 g for 15 min.
FLAG affinity purification
Clarified extracts were incubated with anti-FLAG M2 agarose (Sigma Aldrich) at 4oC for 4 hrs and washed two times with H350 and once with E100 buffer (25 mM HEPES-KOH pH7.5, 10 % glycerol, 100 mM KCl, 2 mM MgCl2, 1 mM EDTA, 0.02% NP40, 1 ug/ml pepstatin A, 2 ug/ml leupeptin, 1 mM PMSF). The FLAG tagged proteins were eluted with E100 buffer with 400 ug/ml 3xFLAG peptide.
Size-exclusion chromatography
Eluates from FLAG purification were loaded onto a Superose 6 size-exclusion column (GE17-5172-01, Amersham Bioscience) equilibrated in superose 6 buffer (350 mM NaCl, 40 mM HEPES pH7.5, 5 % glycerol, 0.1 % Tween 20) for 350 mM NaCl condition. The 500 ul fractions eluted from the column were analyzed by western blots and multidimensional protein identification technology (MudPIT).
Multidimensional Protein Identification Technology
Proteins isolated from FLAG-AP of (Halo-E1-alpha and E1-alpha-Halo) were precipitated with 20% trichloroacetic acid (TCA) and the resulting pellet was washed once with 10% TCA and twice with cold acetone. TCA-precipitated protein pellet was 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 (Promega) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The samples 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. The MS/MS data set was searched using ProLuCID (v. 1.3.3) against a database consisting of 36,628 Homo sapiens NR proteins (NCBI -released June 10, 2016), 193 usual contaminants, and, to estimate false discovery rates (FDRs), 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 to an FDR less than 5% at the peptide and protein levels, using DTASelect v1.9 in combination with swallow, an in-house software.
2020-03-20 | MSV000085126 | MassIVE
Project description:Protein pull-down assay- Mixed erythrocytic stage 3D7 Plasmodium falciparum parasite cultures were collected and lysed using 0.15% saponin for 10 min on ice. After subsequent washes, the parasite pellet was resuspended in 2.5X volume of IP buffer (0.65% Igepal CA-360 (Sigma-Aldrich), 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, 1 mM AEBSF, 5 uM E-64 and EDTA-free protease inhibitor cocktail (Roche)) and lysed by passing through a 26G 1/2-inch needle ten times and sonicated 7 times 10 seconds on/30 seconds off using a probe sonicator. Extracted nuclear protein lysates were incubated for 10 mins at room temperature with DNase I to remove DNA and centrifuged for 10 mins at 14,500 rcf to remove cell debris. Washed Protein A magnetic beads (Pure Proteome) were added to the protein sample and incubated for 1 hour at 4oC to preclear the lysate. Precleared lysate was transferred to a new microcentrifuge tube and split equally for the antibody and no antibody control. The anti-CRWN-like custom antibody was added at a 1:50 ratio and incubated overnight at 4oC. The negative control with no antibody was also incubated overnight. Antibody-protein complexes were recovered using Protein A magnetic beads (Pure Proteome), followed by extensive washes with wash buffer A (1% Triton-X, 1 mM EDTA in 1X PBS), wash buffer B (wash buffer A, 0.5 M NaCl) and wash buffer C (1 mM EDTA, 1X PBS). Proteins were eluted using 0.1 M glycine, pH 2.8 and the eluent was neutralized using 2 M Tris-HCl, pH 8.0. Protein eluates were TCA-precipitated overnight, then washed twice with cold acetone.
Multidimensional Protein Identification Technology- The TCA-precipitated protein pellet was 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 (Promega) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The samples 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. The MS/MS data set was searched using ProLuCID (v. 1.3.3) against a database consisting of 5,530 P. falciparum non-redundant proteins (PlasmoDB-34), 36,628 Homo sapiens NR proteins (NCBI -released June 10, 2016), 193 usual contaminants, and, to estimate false discovery rates (FDRs), 42,351 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 to an FDR less than 5% at the peptide and protein levels, using DTASelect in combination with swallow, an in-house software.
2019-12-10 | MSV000084671 | MassIVE
Project description:Protein pull-down assay- Mixed erythrocytic stage 3D7 Plasmodium falciparum parasite cultures were collected and lysed using 0.15% saponin for 10 min on ice. After subsequent washes, the parasite pellet was resuspended in 2.5X volume of IP buffer (0.65% Igepal CA-360 (Sigma-Aldrich), 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, 1 mM AEBSF, 5 uM E-64 and EDTA-free protease inhibitor cocktail (Roche)) and lysed by passing through a 26G 1/2-inch needle ten times and sonicated 7 times 10 seconds on/30 seconds off using a probe sonicator. Extracted nuclear protein lysates were incubated for 10 mins at room temperature with DNase I to remove DNA and centrifuged for 10 mins at 14,500 rcf to remove cell debris. Washed Protein A magnetic beads (Pure Proteome) were added to the protein sample and incubated for 1 hour at 4oC to preclear the lysate. Precleared lysate was transferred to a new microcentrifuge tube and split equally for the antibody and no antibody control. The anti-SMC3 custom antibody was added at a 1:50 ratio and incubated overnight at 4oC. The negative control with no antibody was also incubated overnight. Antibody-protein complexes were recovered using Protein A magnetic beads (Pure Proteome), followed by extensive washes with wash buffer A (1% Triton-X, 1 mM EDTA in 1X PBS), wash buffer B (wash buffer A, 0.5 M NaCl) and wash buffer C (1 mM EDTA, 1X PBS). Proteins were eluted using 0.1 M glycine, pH 2.8 and the eluent was neutralized using 2 M Tris-HCl, pH 8.0.
Multidimensional Protein Identification Technology- Two biological replicates with two technical replicates each were prepared for ChEP and cytoplasmic control samples at the ring, trophozoite, and schizont stages. Proteins were precipitated with 20% trichloroacetic acid (TCA) and the resulting pellet was washed once with 10% TCA and twice with cold acetone. About 50 ug of the TCA-precipitated protein pellet was 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 (Promega) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The samples 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. The MS/MS data set was searched using ProLuCID (v. 1.3.3) against a database consisting of 5,530 P. falciparum non-redundant proteins (PlasmoDB-34), 36,628 Homo sapiens NR proteins (NCBI -released June 10, 2016), 193 usual contaminants, and, to estimate false discovery rates (FDRs), 42,351 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 to an FDR less than 5% at the peptide and protein levels, using DTASelect in combination with swallow, an in-house software.
2018-06-28 | MSV000082521 | MassIVE
Project description:Mammalian protein extraction and Halo purification
For Halo purification, HEK293T cells at 40% confluency in a 100 mm plate were transfected with 10ug of plasmid containing ORF of PDHA1-HA-Halo in 1X BBS with 125 mM CaCl2. Cells were harvested by scraping in PBS following PBS wash after 24 hours from transfection. For non-transfected cells, cells were harvested at 100% confluency. The cell pellet was resuspended in 1 ml of Hypotonic buffer (50 mM Tris-HCl pH 7.5, 0.1% NP-40) and centrifuged at 4000 rpm for 5 min. The resulting pellet was resuspended in 1 ml of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 % Triton X-100, 0.1 % Na-deoxycholate) and clarified by centrifugation at 12,000 rpm for 30 min. The crude extract in the supernatant was treated with 125 U of benzonase for 20 minutes in 4oC, then clarified by centrifugation at 12,000 rpm for 10 minutes. The clarified extract was either directly loaded onto Superose 6 column or subjected to Halo-purification by adding with 100 ul of Halo beads suspension incubated overnight in 4oC on a rotator. The beads were washed 3 times then subjected for protein elution with 50 U of AcTEV protease (Invitrogen, 12575-015) in PBS buffer by 8 hr incubation in 4oC on a rotator.
Multidimensional Protein Identification Technology
Proteins isolated from Halo-AP of N- and C-terminally tagged human PDHA1 (Halo-E1-alpha and E1-alpha-Halo) were precipitated with 20% trichloroacetic acid (TCA) and the resulting pellet was washed once with 10% TCA and twice with cold acetone. TCA-precipitated protein pellet was 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 (Promega) at 1:100 w/w. The reactions were stopped using formic acid (5% final). The samples 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. The MS/MS data set was searched using ProLuCID (v. 1.3.3) against a database consisting of 36,628 Homo sapiens NR proteins (NCBI -released June 10, 2016), 193 usual contaminants, and, to estimate false discovery rates (FDRs), 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 to an FDR less than 5% at the peptide and protein levels, using DTASelect v1.9 in combination with swallow, an in-house software.
2020-03-20 | MSV000085125 | MassIVE
Project description:Protein Complexes Purification: We generated 293FRT cell lines that stably express FLAG-tagged ICE1 (Interactor of Little Elongation Complex ELL subunit 1; KIAA0947). We extracted nuclear proteins in the presence of Benzonase to digest nucleic acids, including both DNA and RNA, and purified the little elongation complex (LEC) through anti-FLAG affinity purification using anti-FLAG M2 agarose. Cells only expressing the FLAG epitope-tag were analyzed in parallel as negative controls.
A MED26 hypomorphic HEK293T cell line D2G8 was generated by applying multiple small guide RNAs (sgRNAs) complementary to the MED26 gene to induce random sequence insertions and/or deletions. This cell line expresses mutant MED26 lacking the NTD. Mediator was purified from cells expressing either WT MED26 or D2G8 MED26 via its ability to bind the transcriptional activation domain of ATF6-alpha. Proteins were precipitated with 1/5 TCA overnight at 4oC and washed 2x with cold acetone.
Multidimensional Protein Identification Technology: TCA-precipitated protein pellets were with Tris-HCl pH 8.5 8 M urea, followed by addition of TCEP (Pierce) and chloroacetamide (Sigma) to a final concentration of 5 mM and 10 mM, respectively. Proteins were digested using Endoproteinase Lys-C (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 (Promega). Digested peptides 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 (v. 1.3.3) against 36628 non-redundant Homo sapiens proteins (downloaded from NCBI RefSeq 2016-06-10), 193 usual contaminants, and, to estimate false discovery rates (FDRs), 36821 randomized amino acid sequences derived from each NR protein. To account for alkylation by CAM, 57 Da were added statically to the cysteines. To account for oxidation, 16 Da were added as a differential modification to methionines. Peptide/spectrum matches were sorted and selected to an FDR less than 5% at the peptide and protein levels, using DTASelect in combination with swallow, an in-house software.
2019-02-20 | MSV000083465 | MassIVE
Project 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
2019-11-13 | MSV000084579 | MassIVE
Project description:Ingestion of a diet with a restricted Methionine (MR) content results in robust activation of a set of browning genes in IWAT (inguinal white adipose tissue). So far, data were obtained using a mix of elemental amino acids as the dietary protein source, which was replaced in this experiment with casein. To reduce methionine content, casein was oxidized and methionine was added back to a concentration similar to the diets using elemental amino acids as protein source. Results show that mice reduce body weight and adiposity on MR containing oxidized casein similar to MR containing elemental amino acids. Similarly, energy expenditure, as well as food and water intake were increased. The RNAseq experiment was conducted to compare the effect of MR using these two protein sources on the IWAT transcriptome.
2021-06-09 | GSE162917 | GEO