Project description:Proteomic analysis of chick vestibualr hair bundles purified using the twist-off method. See Description.doc. Utricle hair bundles were purified from E20–21 chicks using the twist-off method (Gillespie & Hudspeth J Cell Biol 112, 625-640, 1991; Shin et al. Neuron 53, 371-386, 2007). NuPAGE LDS sample buffer (Invitrogen) with 50 mM dithiothreitol was added to a combined final volume of 80 µl per 100 utricles' worth of bundles; samples were heated to 70°C for 15 min. Epithelial proteins were similarly solubilized with sample buffer. Proteins were separated by running ~1 cm into a NuPAGE 4–12% Bis-Tris gel (1.5 mm x 10 well; one or two lanes per bundle sample); gels were rinsed with water, then stained with Imperial Protein Stain (Thermo Scientific). The 1 cm of gel with separated proteins was manually sliced into six pieces. Gel pieces were transferred to siliconized tubes and washed and destained in 200 µl 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 30 µl of 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h. The DTT solution was removed and the sample alkylated in 30 µl 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 h. The reagent was removed and the gel pieces dehydrated in 100 µl acetonitrile. The acetonitrile was removed and the gel pieces rehydrated in 100 µl 0.1 M ammonium bicarbonate. The pieces were dehydrated in 100 µl acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/µl trypsin (Sigma-Aldrich T6567 proteomics grade, from porcine pancreas, dimethylated) in 50 mM ammonium bicarbonate on ice for 10 min. Any excess enzyme solution was removed and 20 µl 50 mM ammonium bicarbonate added. The sample was digested overnight at 37°C and the peptides formed extracted from the polyacrylamide in two 30 µl aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 15 µl for MS analysis. For the gel slice immediately adjacent to the agarose, peptides were purified away from interfering polymers by SCX. A single experiment's worth of hair bundles or epithelium was analyzed by six LC-MS/MS runs, corresponding to the six gel pieces. The LC-MS/MS system consisted of a Thermo Electron Orbitrap Velos ETD mass spectrometer system with a Protana nanospray ion source, which was interfaced to a reversed-phase capillary column of 8 cm length x 75 µm internal diameter, self-packed with Phenomenex C18 Jupiter of 10 µm particle size. An extract aliquot (7.5 µl) was injected and peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5 µl/min over 1.2 hr. The nanospray ion source was operated at 2.5 kV. The digest was analyzed using the data-dependent capability of the instrument, acquiring—in sequential scans—a single full scan mass spectrum in the Orbitrap detector at 60,000 resolution to determine peptide molecular weights, and 20 product-ion spectra in the ion trap to determine amino acid sequence. MaxQuant version 1.2.2.5 software was used for protein identification and quantitation. The default contaminants file associated with the MaxQuant download was edited to remove entries known to be present in hair bundles (e.g., actin) and to add additional impurities that entered the bundle-purification workflow (keratins, hemoglobins, egg white components). Mass spectrometry data were searched against Ensembl version 66 (released February, 2012) using Andromeda; the Ensembl FASTA file was edited by replacing several sequences with longer or full-length sequences, including actin gamma 1 (NP_001007825.1), actin beta (NP_990849.1), fascin 1 (NP_001171603), fascin 2 (NP_001171209), ATP synthase beta (NP_001026562.1), peptidylprolyl isomerase A (NP_001159798.1), calbindin 2 (NP_990647.1), PDZD7 (XP_003641537.1), espin (XP_417532.3), and CACNA2D2 (XP_427707.3). Protein identifications were reported with an FDR of 1%. If a set of peptides for a protein was identical to or completely contained within that of another protein, MaxQuant groups those proteins together ("redundant groups"); the entry with the largest number of peptides was used to identify the redundant group. Redundant groups that shared more than 20% of their identified peptides were further grouped in our analysis ("shared-peptide groups"); the entry with the greatest intensity associated with it was used to identify the shared-peptide group.

Project description:Cyanophora paradoxa muroplasts were isolated on a Percoll gradient. Muroplasts were fractionated into soluble (stroma), envelope, and thylakoid proteins. Protein samples from envelope membranes, thylakoids, stroma and total muroplasts were separated by 12% SDS-PAGE. Each Protein lanewas cut into 10 slices of variable size to match similar protein amounts. The gel slices were washed and diced to 1mm3 cubes. Gel pieces were destained by two consecutive washes with a 50:50 mixture of 200 mM ammonium bicarbonate and acetonitrile (ACN) for 20 min at 37°C. Proteins were reduced with 10mM DTT for 45 min at 56°C, followed by alkylation with 55mM iodoacetamide for 45 min at room temperature in the dark. After reduction and alkylation, gel pieces were washed twice with 100 mM ammonium bicarbonate, dehydrated with 100% ACN for 10 min, and dried in a SpeedVac. Gel pieces were fully covered in 50 mM ammonium bicarbonate containing 10ng/ml trypsin and rehydrated at 4°C. The samples were incubated overnight at 37°C and peptides in the supernatant were pooled after successive extraction steps using 50 mM ammonium bicarbonate, 100% ACN and 2.5% formic acid (FA), 50% ACN. The samples were dried in a SpeedVac and reconstituted in 5% ACN, 0.1% FA prior to MS analysis. Each of the samples were analysed in duplicates. Tryptic peptides of the thylakoid, envelope and stroma fractions were loaded onto a fritless 100 μm capillary packed in-house with 10cm of ProntoSIL C18 ace-EPS, 3µm. A gradient of 5-80% (v/v) ACN in 0.1% (v/v) FA was passed over the column with a duration of 115 min. The eluted peptides were directly electrosprayed into the LTQ orbitrap XL MS at a flow rate of 250 nl min-1 and a spray voltage of 1.3 kV. The instrument was operated in a 4-step data-dependent positive mode to identify the top three most abundant ions in a high-resolution MS scan (400 to 2000 m/z), which were then selected for fragmentation. MudPIT analyses were performed on the muroplast samples using an in-house packed analytical column consisting of 10 cm ProntoSIL C18 ace-EPS, 3µm (Bischoff) and 2 cm of a strong cationic exchange (SCX) material, 3µm in combination with an 6-step salt pulse gradient (0, 50, 100, 150, 200 and 500 mM ammonium acetate). Each salt-step peptides were eluted from the SCX to the C18-phase of the analytical column and eluated with a 130- min reverse-phase solvent gradient (5–80% ACN containing 0.1% FA). The eluate was directly electrosprayed into the LTQ orbitrap XL MS which was operated as described before. All MS/MS samples were analyzed using Sequest and X! Tandem. Sequest was set up to search the C. paradoxa nuclear and muroplast protein database with a total of 32,286 entries, assuming the digestion enzyme trypsin. X! Tandem was set up to search a subset of the C. paradoxa database also assuming trypsin. Sequest and X! Tandem were searched with a fragment ion mass tolerance of 0,80 Da and a parent ion tolerance of 10,0 ppm. Oxidation of methionine and iodoacetamide derivatives of cysteine were specified in Sequest and X! Tandem as variable modifications. Scaffold (version 3.5.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95,0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99,0% probability and contained at least 2 identified peptides.

Project description:Some molecular and cellular mechanisms of fertilization are still not completely described. The role of the acrosomal matrix (AM), the particulate compartment within the acrosome, is one of them. Here, we proposed that amyloids, highly ordered protein aggregates, by their physical and chemical properties could contribute to AM roles in fertilization. Purified AM were exposed to a two-step extraction to sequentially strip off soluble proteins. The remaining insoluble material (core) was subjected to a mass spectrometry analysis. Preparation of samples for MS analysis: Three different approaches were used to optimize identification of peptides in the AM core. For in-gel digestion, core samples were resuspended in 13.2mM SA pH3 containing 8M urea and 100 mM DTT and incubated for 1 hr at RT. Proteins were separated on a hand casted 12% polyacrylamide Tris-glycine gel. After silver staining, visible bands were cut from the gel, destained and washed with ddH2O (2 X 5 min) then with 50% ethanol (2 X 5 min) before stored at -20 degrees C. Gel pieces were tryptically digested in 25mM triethylammonium bicarbonate buffer at 37 degrees C overnight with trypsin. For in-solution digestion, core samples were resuspended in 13.2mM SA pH3/ 8M urea/ 100mM DTT and incubated for 1 h at RT followed by 15 min at 70 degrees C. Iodoacetamide was added to 10mM and proteins were alkylated by incubation for 15 min at RT in the dark. Samples were added to a pre-rinsed spin filter and centrifuged at 14,000 X g. Samples were washed with 9M urea then with 25mM ammonium bicarbonate. Samples were digested with trypsin in 25mM ammonium bicarbonate overnight. After digestion, samples were spin at 14,000 X g and washed 2 times with 25mM ammonium bicarbonate. The retentate was transferred to a new tube and air dried. For on-membrane digestion, the samples were dotted on nitrocellulose membrane and digested by trypsin. Briefly, core samples were resuspended and treated as for in-solution digestion. Then, samples were dotted by gravity on the membrane. Wells were rinsed with 20mM SA pH3 followed by TBS. Dots were cut and air-dried. After the protein digestion with trypsin in 25mM ammonium bicarbonate the membranes were dissolved with acetone and the precipitated peptides were air-dried. All digested peptides were reconstituted in 2% acetonitrile/ 0.1% formic acid for MS analysis. MS data acquisition: Protein identification by liquid chromatography tandem mass spectrometry (LCMS/MS) analysis of peptides was performed using an LTQ Orbitrap Velos MS interfaced with a 2D nanoLC system. Protein and Peptide Identification: MS/MS spectra were extracted using the ProteoWizard Toolkit. The spectra were analyzed using the GPM Manager with X!Tandem to search against a home made mouse database containing 213,054 nonredundant protein sequences created by using mouse sequences from Ensembl database (files Mus_musculus.GRCm38.73.pep.all & Mus_musculus.GRCm38.73.pep.abinitio) and from NCBI database (file nr downloaded on 09/19/2013) . Search was performed using fully tryptic enzyme specificity (See deposited MS data for details). Peptides and proteins that have an expectation value of log10 (e) <= -2 were included in the results. Curated results were obtained by keeping only proteins with at least one mouse-specific matching peptide (peptide match = 100% identities and 100% coverage on a unique mouse protein and <100% identities and/or <100% coverage on human proteins). In addition, trypsin-like proteins were kept if at least one peptide was not matching on exogenous pig trypsins.

Project description:Data analysis. Spot detection and matching were performed with a comparative cross analysis of all the gels using DeCyder software v.6.5 (GE Healthcare). 178 spots were selected based on 1.15-fold for protein ratio cut-off, allowing for the appearance of the spots in 23 out of 28 gels (69 out of 84 total images). Data from 95 spots were submitted. 93 spots were identified with high confidence. Spot picking and Trypsin digestion. The spots of interest were picked up by Ettan Spot Picker (GE Healthcare) based on the in-gel analysis and spot picking design by DeCyder software. The gel spots were washed a few times then digested in-gel with modified porcine trypsin protease (Promega, Fitchburg, WI). The digested tryptic peptides were desalted using a Zip-tip C18 (Millipore, Billerica, MA). Peptides were eluted from the Zip-tip with 0.5 uL of matrix solution (alpha-cyano-4-hydroxycinnamic acid, 5 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid, 25mM ammonium bicarbonate) and spotted on a MALDI plate. Mass Spectrometry. MALDI-TOF MS and TOF/TOF tandem MS/MS were performed on AB SCIEX TOF/TOF 5800 System (AB SCIEX). MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 4000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 4000 laser shots per fragmentation spectrum on each of the 7-10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions). Database search. Both the resulting peptide mass and the associated fragmentation spectra were submitted to GPS Explorer workstation equipped with MASCOT search engine (Matrix Science, Boston, MA) to search the Swiss-Prot database. Searches were performed without constraining protein molecular weight or isoelectric point, with variable carbamidomethylation of cysteine and oxidation of methionine residues, and with one missed cleavage also allowed in the search parameters. Candidates with either protein score C.I.% or Ion C.I.% greater than 95 were considered significant. When multiple IDs were significant for a given spot, the selection was made by evaluating apparent molecular weight, isoelectric point, the location of the spot in the gel, and the presence of strips of multiple protein isoforms in the adjacent spots.

Project description:Mitochondrial fraction was obtained using the mitochondrial isolation kit (KC010100, BioChain Institute). Each tissue was finely chopped and homogenized 40 strokes in two volumes of mitochondrial isolation buffer, and then centrifuged at 600×g for 10 min. The supernatant was collected and centrifuged at 12,000×g for 15 min. The supernatant was used as cytosolic fraction containing lysosomes and microsomes. The mitochondrial fraction was obtained from the pellet and resuspended in mitochondrial isolation buffer. The suspended mitochondria were sonicated (TOMY, Ultrasonic disruptor UD-100, output level 30, 30 sec×2) and centrifuged at 105,000×g for 30 min. The supernatant was used as the mitochondrial soluble fractions, containing mitochondrial intermembrane space (IMS) and matrix (MAT). The pellet was resuspended in RIPA buffer and centrifuged at 20,000×g for 30 min. The supernatant was used as the mitochondrial membrane fractions, containing mitochondrial outer membrane (OM) and inner membrane (IM). All the procedures were performed at 4°C. Mitochondrial and cytosolic fractions of WT and ES1-KO mouse brain were isolated as described 2.1 in 1% triton x-100 in 20 mM Tris-HCl. These samples were concentrated by SDS-PAGE and cut out from gels. The sectioned gels were finely chopped and washed with water. Then, the gel pieces were destained 50% methanol and vortexed in 25 mM ammonium bicarbonate containing 50% acetonitrile for 5 min. The supernatant was removed and the gel pieces were soaked 100% acetonitrile for 1 min. After removing the solution, the gel pieces were dried in vacuo, and rehydrated with a reducing solution (50 mM ammonium bicarbonate and 25 mM dithiothreitol), and incubated for 40 min at 56°C. The reducing buffer was removed and gel pieces were soaked in an alkylating solution (50 mM ammonium bicarbonate containing 55 mM iodoacetamide) for 30 min at room temperature in the dark. After removing the alkylation solution, the gel pieces were washed twice with water and vortexed in 25 mM ammonium bicarbonate containing 50% acetonitrile for 5 min, and then soaked 100% acetonitrile for 1 min. After removing the solution, the gel pieces were dried in vacuo and rehydrated with 50 mM ammonium bicarbonate containing 4 ng/μL sequencing grade trypsin and 0.01% Protease Max Surfactant. After incubating 10 min on ice, an equivalent volume of 50 mM ammonium bicarbonate containing 0.01% Protease Max Surfactant was added and incubated for 3 h at 37°C to digest the proteins in the gels with a protease. The equivalent volume of 1% trifluoroacetic acid was added and vortexed for 10 min for extraction of the tryptic fragments. After centrifuged at 12,000×g for 15 min, the peptide solutions were obtained from the supernatant. Ten microliters of each peptide solution were diluted with 190 μL of 100 mM ammonium bicarbonate and filtered on with a 0.22 μm using the amicon ultrafiltration unit (Amicon Ultra, 3 kDa, Millipore, MA, USA). Concentrated samples were denatured with an equivalent volume of trifluoroethanol (25 μL) and reduced with 1 μL of 200 mM dithiothreitol (DTT). The samples were incubated at 90°C for 30 min and cooled to room temperature. Free cysteine residues were alkylated with 4 μL of 200 mM iodoacetamide for 60 min at room temperature in the dark and the remaining iodoacetamide was quenched by adding 1 μL of 200 mM DTT. The samples were then mixed with 300 μL of 100 mM ammonium bicarbonate. Fifteen microliters of the sample were diluted with 85 μL of 100 mM ammonium bicarbonate and incubated with 1 μg trypsin (TPCK treated, AB Sciex, Framingham, MA, USA) at 37 °C for 18 h. The samples were desalted with C18 ZipTip (Millipore, Bedford, MA, USA) and eluted with H2O/acetonitrile (5/5; v/v). The ZipTip eluates were dried in a vacuum centrifuge. Desalted samples were rehydrated in 0.1% formic acid (FA) and were analyzed by LC-MS using a nanoLC Eksigent 400 system (Eksigent, AB Sciex), coupled online to a TripleTOF6600 mass spectrometer (AB Sciex). Peptide separation was performed using liquid chromatography with a trap and elution conﬁguration using a nano trap column (350 μm×0.5 mm, 3 μm, 120 Å, AB Sciex) and a nano ChromXP C18 reverse-phase column (75 μm×15 cm, 3 μm, 120 Å, AB Sciex) at 300 nL/min with a 90 min linear gradient of 8-30% acetonitrile in 0.1% FA, and then, with a 10 min linear gradient of 30% to 40% acetonitrile in 0.1% FA. The mass spectrometer was operated in information-dependent acquisition (IDA) mode, scanning full spectra (400–1500 m/z) for 250 ms, followed by up to 30 MS/MS scans (100–1800 m/z for 50 ms each), for a cycle time of 1.8 s. Candidate ions with a charge state between +2 and + 5 and counts above a minimum threshold of 125 counts per second were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 12 s. The rolling collision energy was used with a collision energy spread of 15. The mass spectrometer was operated using the Analyst TF 1.7.1 software program (AB Sciex). For data-dependent acquisition (DDA, SWATH acquisition), the parameters were set as follows: 100 ms TOF MS scan, followed by 200 variable SWATH windows each at 50 ms accumulation time for m/z 400–1250. MS/MS SWATH scans were set at 5 Da window overlapping by 1 Da for m/z 400–1250 and varied on each side of the mass range. The total cycle time was 9.6 s. Rolling collision energy (CE) parameter script was used to automatically control the CE. Acquired spectra were searched against the UniProt reviewed database using the Paragon algorithm embedded in the ProteinPilot 5.0.1 software program (AB Sciex), with the following search parameters: (i) sample type: identiﬁcation, (ii) Cys alkylation: iodoacetamide, (iii) digestion: trypsin, (iv) instrument: TripleTOF 6600, (v) species: Mus musculus, (vi) ID focus: biological modiﬁcations, (vii) detected protein threshold: > 0.05 (10% conﬁdence). The detected protein threshold was set to the minimum level to enhance the number of wrong answers to enable the curve ﬁtting by an independent FDR analysis. This was carried out by the target-decoy approach provided with the ProteinPilot software program, which was used to assess the quality of the identiﬁcations. Positive identiﬁcations were considered when identiﬁed proteins and peptides reached a 1% local FDR. The resulting group file was loaded into Peakview (v2.2.0, AB Sciex) and peaks from SWATH runs were extracted with a peptide confidence threshold of 99% and a false discovery rate <1%. The SWATH ﬁles were then exported to the MarkerView software program (version 1.3.0.1; AB Sciex) and the peak areas of individual peptides were normalized to the sum of the peak areas of all detected peptides.

Project description:Using cell sorting technique, we collected Arabidopsis root hair protoplasts (transgenic line harboring root hair specific gene promoter ::GFP, here named GFP) and nonGFP protoplasts( whole root except root hair). Then, we extracted total proteins and RNA for proteome and RNASeq. RNAseq data have already been deposited into NCBI (accession numbers SRR364677 and SRR364678). Total proteins were separated by 1D SDS-PAGE, followed by gel slicing, in gel digested and run ESI-LC-MS/MS on LTQ Orbitrap Velos. We set two biological repeats and each sample was scanned two times (two technique repeats). We then combined all ms/ms raw data from all fractions and technique repeats from one biological sample into one file to indentify proteins by searching Arabidopsis protein database with software Proteome Discoverer (with two searching engines mascot and sequester). Protocol: Protoplasts from EXP7 and non-GFP protoplastswere stored at -80°C, thawed on ice for 5 min, vortexed several times and suspended in 5 x volume of pre-cooled acetone (-20°C) containing 10% (v/v) trichloroacetic acid (TCA) and 0.07% (v/v) 2-mercaptoethanol. Proteins were then precipitated for 2 h at −20°C after thorough mixing. Proteins were collected by centrifuging at 35,000 g (JA-20 108 rotor, Beckman J2-HS) at 4°C for 30 min. The supernatant was removed and the protein pellets were washed three times with cold acetone containing 0.07% (v/v) 2-mercaptoethanol and 1 mMphenylmethanesulfonylfluoride. Protein pellets were dried by lyophilization and stored at -80°C, or immediately extracted using Laemmli buffer (63 mM TrisHCl pH 6.8, 10% glycerol, 2% SDS, 0.0025% bromophenol blue). Protein concentration was determined using Pierce protein assay kit (Pierce, Rockford). Proteins from each sample were separated using a Bis-Tris precast gradient gel (4−16%, Bio-Rad) according to the manufacturer’s instructions. After electrophoresis, the gel was Coomassie-stained using the Colloidal Blue Staining Kit (Invitrogen). The protocol used for in-gel trypsin digestion of proteins in gels was adapted from the method described in Shevchenko et al. (1996). Briefly, protein bands were manually excised and each band was cut into small pieces (~0.5 mm). The gel pieces were washed three times with a solution containing 50% methanol and 5% acetic acid for 2 h, twice with a solution containing 25 mM NH4HCO3 in 50% acetonitrile for 10 min each, and then the gel pieces were dried in a vacuum centrifuge. Reduction of proteins in gel pieces was performed with DTT and alkylation with iodoacetamide, and the gel pieces were washed and dried in a vacuum centrifuge. A trypsin solution in 25 mM NH4HCO3 containing 75 to 100 ng of sequencing grade modified trypsin (Promega) in 25-40 µl was added and incubated with gel pieces for 12-16 h at 37°C. To recover the tryptic peptides, a solution of 30 µl containing 5% formic acid and 50% acetonitril was added to the gel pieces, agitated in a vortex for 30-60 min and withdrawn into a new tube. The recovery was repeated once with 15 µl solution and the resulting two recovers were combined and dried in a vacuum centrifuge. The dried pellet was re-dissolved in 10-20 µl of 0.1% formic acid for LC-MS/MS analysis.

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Mass spectrometry analysis of ABHD17B partners in hepatic stellate cells

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