Project description:Proto-myofibroblasts were treated with siRNA, washed with ice-cold HEPES-saline (20 mM HEPES-NaOH [pH 7.5], 137 mM NaCl), detached by Accutase (Nacalai Tesque), and lysed in guanidine-TCEP buffer (8 M guanidine-HCl, 100 mM HEPES-NaOH [pH 7.5], 10 mM TCEP, 40 mM CCA). After heating and sonication, 200 Î¼g of protein was purified by methanol-chloroform precipitation and resuspended in 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. Proteins were further sonicated, heated, and digested with trypsin/Lys-C mix (Promega) for 16 hours at 37Â°C. Peptide concentrations were determined using the Pierce quantitative colorimetric peptide assay (Thermo Fisher Scientific). Approximately 130 Î¼g of peptides was labeled with TMTpro 16-plex reagent (Thermo Fisher Scientific) for 1 hour at 25Â°C. After quenching with hydroxylamine, labeled samples were pooled. One-tenth of the pooled sample was then acidified with trifluoroacetic acid (TFA) and fractionated by offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC system (Thermo Fisher Scientific). The 48 fractions were consolidated into 16 fractions, evaporated in a SpeedVac concentrator, and disolved in 3% acetonitrile (ACN) and 0.1% TFA. LC-MS/MS analysis of the resulting peptides (500 ng each) was performed on an EASY-nLC 1200 UHPLC coupled to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were separated on a 75 Î¼m inner diameter Ã— 150 mm C18 reversed-phase column (Nikkyo Technos) using a linear gradient of 4%â€“20% ACN for 0â€“115 minutes and 20%â€“32% ACN for 115â€“160 minutes, followed by an increase to 80% ACN for 10 minutes and a final 10-minute hold at 80% ACN. The mass spectrometer was operated in data-dependent acquisition mode with a top-10 MS/MS method. MS1 spectra were acquired at a resolution of 70,000, an automatic gain control (AGC) target of 3e6, and a mass range of 375â€“1400 m/z. HCD MS/MS spectra were acquired at a resolution of 35,000 with an AGC target of 1e5, an isolation window of 0.7 m/z, a maximum injection time of 150 ms, and a normalized collision energy of 33. Dynamic exclusion was set to 20 seconds. Raw data were analyzed against the Mus musculus SwissProt database using Proteome Discoverer version 2.5 (Thermo Fisher Scientific) with the Sequest HT search engine for protein identification and TMT quantification. Search parameters included trypsin with up to 2 missed cleavages, a precursor mass tolerance of 10 ppm, and a fragment mass tolerance of 0.02 Da. TMTpro labeling of lysine residues and peptide N-termini and carbamidomethylation of cysteine were set as fixed modifications, and methionine oxidation was set as a variable modification. Peptides and proteins were filtered at a false discovery rate (FDR) of 1% using the percolator node and protein FDR validator node, respectively. Normalization was performed so that the total abundance across all peptides was equal for each TMT channel.

Project description:Female Sprague-Dawley rats were randomly separated into two groups: i) trained (Ex group) and ii) sedentary (Cont group). Animals from the Ex group were adapted to treadmill exercise for two consecutive weeks, involving a gradual increase in the running time till 60 min/day at 20 m/min, 0% grade, 5 days/week. This exercise program remained constant until the end of the 54 weeks. Mitochondria isolation from 10 animals (5 Ex group and 5 Cont group) was performed using the conventional methods of differential centrifugation. Mitochondrial protein extracts were reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin using the FASP procedure (18). Briefly, each sample was solubilized in 4 % SDS, 0.1 M DTT, 0.1 M HEPES and incubated for 30 min at 60 C. Then, samples were mixed with 0.2 mL of a solution of 8 M Urea, 0.1 M HEPES, pH 8.5 (UH solution), loaded into the filtration devices (3k Microcon, Millipore), centrifuged at 14,000 g for 20 min, and washed twice with UH solution. After centrifugation, the concentrates were alkylated with 50 mM iodoacetamide in UH solution (dark, 25 C, 20 min), and washed twice with UH solution. The concentrate was diluted with 0.1 mL of 50 mM ammonium bicarbonate (ABC) and digested overnight (37 C) with the addition of 2 ug of trypsin diluted in 30 uL of 50 mM ABC. The resulting tryptic peptides were collected by centrifugation and the filter were washed twice with 50 uL of 50 mM ABC. Eluted peptides were acidified with 10 uL of formic acid and cleaned up on a homemade Empore C18 column (3M, St. Paul, MN, USA) (ref) for subsequent phospho-enrichment and/or analysis by LC-MS/MS. The dried protein digest was dissolved with 10 uL (3 % ACN, 0.1 % TFA), diluted 1:5 with loading buffer (1 M glycolic acid, 5 % TFA, 80 % ACN) and phosphopeptides were enriched on TiO2-beads as previously described (19). Briefly 5 mg of "Titansphere TiO2 5 um" were suspended in 100 uL of 100 % ACN, immobilized in a pipette-column and washed using 5 uL of loading buffer. Each sample was loaded in each pipette-column and then washed with 30 uL of washing buffer (1 % TFA, 80 % ACN). Phosphopeptides were eluted from the beads with 25 uL of 25 % ACN (v/v) containing 25 % NH4OH (m/v), acidified with 10 uL of 10 % TFA and vacuum- concentrated to approximately 4 uL. Samples were finally diluted to 10 uL with H2O + 0.1% formic acid prior to injection. The peptides mixtures were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) on an EASY-nLC system (Proxeon Biosystems, Odense, Denmark) connected to the LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, Bremen, Germany) through a nanoelectrospray ion source. Six microliters of the peptide mixture were auto-sampled directly onto the analytical HPLC column (120 mm x75 um I.D., 3 um particle size (Nikkyo Technos Co., Ltd.) with a 120-min gradient from 5 % to 50 % ACN in 0.1 % FA. The effluent from the HPLC column was directly electrosprayed into the mass spectrometer. The LTQ Orbitrap Velos instrument was operated in data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The MS survey scan was performed in the FT cell recording a window between 350 and 2000 m/z. The resolution was set to 60 000, and the automatic gain control was set to 1,000,000 ions with a maximal acquisition time of 400 ms. The 20 most intense peptide ions with charge states great than or equal to 2 were sequentially isolated to a target value of 5,000 and fragmented in the high-pressure linear ion trap by low-energy CID with normalized collision energy of 35% Q value of 0.25, and an activation time of 10 ms. Raw files were processed using Proteome Discoverer version 1.3 (Thermo Fisher Scientific, Bremen). Peak lists were searched using Mascot software version 2.3 (Matrix Science, UK) against a SwissProt database containing entries corresponding to Rattus norvegicus (version of July 2012), a list of common contaminants, and all the corresponding decoy entries. Trypsin was chosen as enzyme and a maximum of two miscleavages were allowed. Carbamidomethylation (C) was set as a fixed modification, whereas oxidation (M) and phosphorylation (STY) were used as variable modifications. Searches were performed using a peptide tolerance of 7 ppm, a product ion tolerance of 0.5 Da. Resulting data files were filtered for FDR less than 1 % at peptide level.

Project description:Prefrontal cortex tissue slices from a cognitively and neurpathologically normal human subject were obtained from the University of Pennsylvania (UPenn) Brain Bank. 300-350 mg grey matter was homogenized in 1.5 ml solution A (0.32 M sucrose, 1mM MgCl2 and 0.1mM CaCl2) with a Teflon pestle. ~100 ?l of the homogenate was saved, solubilized with 1% SDS and clarified by centrifugation. To prepare the [13C6]brain ISTD, 400 mg labeled MouseExpress brain tissue (Cambridge Isotopes, MA) was homogenized in 4 ml buffer A as described above, centrifuged at 1,000 x g for 10 min to clarify, agitated on a rocker at 4�C for 30 min, and centrifuged at 10,000 x g for 20 min. The pellet, a crude membrane fraction, was dissolved in 2 ml 20 mM Tris pH 7.4 with 1% SDS. Total protein in all preparations was quantified with the micro BCA assay (Pierce) and all solutions were supplemented with protease and phosphate inhibitor cocktails (Sigma) as well as 1 mM sodium fluoride and sodium orthovanadate (Sigma). Human cortex homogenate was mixed with the [13C6]brain ISTD (1?g/?l) at a ratio of 2:1(?g/?g) and processed for LC-MS/MS: 40 ?l preparations were heated with lithium dodecyl sulfate (LDS) (Invitrogen) buffer at 95�C for 20 min, separated on a 1.5 mm 4-12% Bis-Tris Gel (Invitrogen), cut into five fractions, chopped into ?2 mm cubes, washed in 200 ?l 50% acetonitrile (ACN) containing 25 mM NH4HCO3, reduced in 300 ?l 10 mM DTT, alkylated in 300 ?l 55 mM iodoacetamide (Sigma), and digested with 80-120 ?l trypsin (.025 ?g/?l) (Promega) overnight at 37�C, so that the amount of trypsin was 1:5 of the total protein to be digested by mass. Peptides were recovered from gel cubes into 200 ?l 50/50 H2O/ACN with 3% formic acid by vortex-mixing and sonication for 20 min each, twice. Samples were then evaporated to a 100 ?l volume, brought to 1ml in H20 with 0.1% formic acid, desalted with Oasis� HLB cartridges (Waters), evaporated almost to completion and suspended in ~ 45?l H2O with 0.1% formic acid, and filtered with 0.22 �m Ultra free-MC filter cartridges (Millipore). LC-data dependant -MS/MS analyses were conducted on a Q Exactive (ThermoFisher Scientific) quadrupole orbi-trap hybrid instrument with an Easy nLC-II (ThermoFisher Scientific) nano-pump/autosampler. 3?l peptide sample (~ 1 ug) was loaded and resolved on a 20 cm x 75 �m proteopepII C18 packed tip column over a 90 min gradient at a flow rate of 350 nL/min, 0-35% mobile phase B (ACN, 0.1 formic acid). A data-dependent top 10 method was used to acquire a 70,000 resolution full scan to trigger ten 17,500 resolution HCD scans. Ions were isolated for MS/MS analysis with a 2.0 Da window on the quadrupole. Average cycle times were 1.2 sec. Each sample was analyzed in triplicate. Raw files from triplicate injections were searched together within Proteome Discoverer 1.3 (ThermoFisher Scientific) using SEQUEST and the human refseq. database (release 47, ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/). Search parameters allowed trypsin to cleave after Lysine and Arginine and have 2 missed cleavages. Precursor ion mass tolerance was set to 15 ppm and fragment ion mass tolerance was 20 mmu. The dynamic modification of methionine (oxidation = 15.995 Da) and lysine (13C6 = 6.020 Da), in addition to the static modification of cysteine (carbamidomethylation = 57.021 Da) was accepted on up to 4 residues per peptide. Within the Proteome Discoverer Software, SILAC pairs were identified using the �2 plex� workflow node, and all peptides were rescored using the Percolator algorithm node. Finally, peptides were filtered at 1% false discovery rate (FDR).

Project description:ΔB-Raf:ER cells were treated with 10 µM U0126 or 1 µM 4-HT for 30 min. The cells were lysed in 6 M guanidine-HCl containing 100 mM HEPES-NaOH (pH7.5), 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication and then centrifuged. The supernatants were recovered, and proteins (200 µg each) were purified by methanol-chloroform precipitation and solubilized with 40 µL of 0.1% RapiGest SF in 50 mM TEAB. After sonication and heating, the proteins were digested with 2 µg trypsin/Lys-C mix (Promega) at 37 °C overnight. The resultant peptides were subjected to the High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific), and the eluates were desalted using GL-Tip SDB (GL Sciences). The desalted eluates were evaporated in a SpeedVac concentrator and redissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source. The peptides were separated on a 75-µm inner diameter × 150-mm C18 reversed-phase column (Nikkyo Technos) with a linear 4–32% ACN gradient for 0–160 min followed by an increase to 80% ACN for 10 min and finally hold at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a top 10 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an AGC target of 1e6, and a mass range from 350 to 1,500 m/z. MS/MS spectra were acquired at a resolution of 17,500, an AGC target of 5e4, an isolation window of 2.0 m/z, a maximum injection time of 60 ms, and a normalized collision energy of 27. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the Swiss-Prot database restricted to Mus musculus using Proteome Discoverer 2.4 with the Sequest HT search engine. The search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.02 Da; (d) carbamidomethylation of cysteine as a fixed modification; (e) acetylation of protein N-terminus, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine as variable modifications. Peptides were filtered at FDR of 1% using the Percolator node of Proteome Discoverer. Label-free quantification was performed based on intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.

Project description:Brain tissues were homogenized in 6 M guanidine-HCl containing 100 mM HEPES−NaOH (pH 7.5), 10 mM Tris (2-carboxyethyl) phosphine (TCEP), and 40 mM chloroacetamide. After heating and sonication, proteins were purified by methanol−chloroform precipitation and solubilized with 0.1% 21 RapiGest SF in 50 mM triethylammonium bicarbonate. The proteins were digested with trypsin/Lys-C mix (Promega) at 37 ºC overnight. 250 µg of digested peptides from each sample were labeled with TMT 10plex reagents (Thermo Fisher Scientific). Labeled peptides were enriched using a High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific). The labeled peptides were pooled and fractionated using a Pierce High pH reversed-phase peptide fractionation kit (Thermo Fisher Scientific). The labeled peptides were pooled and fractionated using offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC system (Thermo Fisher Scientific). Chromatographic separation was performed on a C18 reversed-phase column (4.6 × 250 mm Xbridge BEH130 C18 column with 3.5 mm particles; Waters) with a multistep gradient of mobile phase A (10 mM ammonium formate at pH 9.0 in 2% ACN) and B (10 mM ammonium formate pH 9.0 in 80% ACN). Peptides were separated into 48 fractions, which were consolidated into 16 fractions. Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA. LC-MS/MS analysis was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a C18 reversed-phase column (75 μm × 150 mm; Nikkyo Technos) with a linear gradient of 4−20% for 0−115 min and 20−32% for 115−160 min, followed by an increase to 80% ACN for 10 min and finally a hold at 80% ACN for 10 min. The mass spectrometer was operated in datadependent acquisition mode with a top 10 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an AGC target of 3e6, and a mass range from 375 to 1400 m/z. MS/MS spectra were acquired at a resolution of 35,000, an AGC target of 1e5, an isolation window of 0.7 m/z, a maximum injection time of 150 ms, and a normalized collision energy of 34. Dynamic exclusion was set to 20 s. Raw MS data were searched against the SwissProt database restricted to Mus musculus using Proteome Discoverer version 2.4 with the Sequest HT search engine. Analysis of MS spectra was performed using the following parameters: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.02 Da; (d) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; and (e) phosphorylation of serine, threonine and tyrosine and oxidation of methionine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node.

Project description:Proto-myofibroblasts were treated with siRNA, washed with ice-cold HEPES-saline (20 mM HEPES-NaOH [pH 7.5], 137 mM NaCl), detached by Accutase (Nacalai Tesque), and lysed in guanidine-TCEP buffer (8 M guanidine-HCl, 100 mM HEPES-NaOH [pH 7.5], 10 mM TCEP, 40 mM CCA). After heating and sonication, 200 Î¼g of protein was purified by methanol-chloroform precipitation and resuspended in 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. Proteins were further sonicated, heated, and digested with trypsin/Lys-C mix (Promega) for 16 hours at 37Â°C. Peptide concentrations were determined using the Pierce quantitative colorimetric peptide assay (Thermo Fisher Scientific). Approximately 130 Î¼g of peptides was labeled with TMTpro 16-plex reagent (Thermo Fisher Scientific) for 1 hour at 25Â°C. After quenching with hydroxylamine, labeled samples were pooled. Nine-tenths of the pooled sample were subjected to phosphopeptide enrichment using the High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific). The enriched phosphopeptides were subsequently fractionated into 16 fractions by offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC system (Thermo Fisher Scientific), evaporated in a SpeedVac concentrator, and dissolved in 3% acetonitrile (ACN) and 0.1% TFA. LC-MS/MS analysis of the resulting peptides was performed on an EASY-nLC 1200 UHPLC coupled to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were separated on a 75 Î¼m inner diameter Ã— 150 mm C18 reversed-phase column (Nikkyo Technos) using a linear gradient of 4%â€“20% ACN for 0â€“115 minutes and 20%â€“32% ACN for 115â€“160 minutes, followed by an increase to 80% ACN for 10 minutes and a final 10-minute hold at 80% ACN. The mass spectrometer was operated in data-dependent acquisition mode with a top-10 MS/MS method. MS1 spectra were acquired at a resolution of 70,000, an automatic gain control (AGC) target of 3e6, and a mass range of 375â€“1400 m/z. HCD MS/MS spectra were acquired at a resolution of 35,000 with an AGC target of 1e5, an isolation window of 0.7 m/z, a maximum injection time of 150 ms, and a normalized collision energy of 33. Dynamic exclusion was set to 20 seconds. Raw data were analyzed against the Mus musculus SwissProt database using Proteome Discoverer version 2.5 (Thermo Fisher Scientific) with the Sequest HT search engine for protein identification and TMT quantification. Search parameters included trypsin with up to 2 missed cleavages, a precursor mass tolerance of 10 ppm, and a fragment mass tolerance of 0.02 Da. TMTpro labeling of lysine residues and peptide N-termini and carbamidomethylation of cysteine were set as fixed modifications, and methionine oxidation and serine/threonine/tyrosine phosphorylation were set as a variable modifications. Peptides were filtered at a false discovery rate (FDR) of 1% using the percolator node. Normalization was performed so that the total abundance across all peptides was equal for each TMT channel.

Project description:Mice brain synaptosomal fractions were homogenized in 6 M guanidine-HCl containing 100 mM HEPES−NaOH (pH 7.5), 10 mM Tris (2-carboxyethyl) phosphine (TCEP), and 40 mM chloroacetamide. After heating and sonication, proteins were purified by methanol−chloroform precipitation and solubilized with 0.1% 21 RapiGest SF in 50 mM triethylammonium bicarbonate. After sonication and heating at 95 ºC for 10 min, the proteins were digested with trypsin/Lys-C mix (Promega) at 37 ºC overnight. 20 µg of digested peptides from each sample were labeled with 0.1 mg of TMTpro 16-plex reagents (Thermo Fisher Scientific). The labeled peptides were pooled and fractionated using offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC system (Thermo Fisher Scientific). Chromatographic separation was performed on a C18 reversed-phase column (4.6 × 250 mm Xbridge BEH130 C18 column with 3.5 mm particles; Waters) with a multistep gradient of mobile phase A (10 mM ammonium formate at pH 9.0 in 2% ACN) and B (10 mM ammonium formate pH 9.0 in 80% ACN). Peptides were separated into 48 fractions, which were consolidated into 16 fractions. Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA. LC-MS/MS analysis was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a C18 reversed-phase column (75 μm × 150 mm; Nikkyo Technos) with a linear gradient of 4−20% for 0−115 min and 20−32% for 115−160 min, followed by an increase to 80% acetonitrile (ACN) for 10 min and finally a hold at 80% ACN for 10 min. The mass spectrometer was operated in datadependent acquisition mode with a top 10 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an AGC target of 3e6, and a mass range from 375 to 1400 m/z. MS/MS spectra were acquired at a resolution of 35,000, an AGC target of 1e5, an isolation window of 0.7 m/z, a maximum injection time of 150 ms, and a normalized collision energy of 33. Dynamic exclusion was set to 20 s. Raw MS data were searched against the SwissProt database restricted to Mus musculus using Proteome Discoverer version 2.4 with the Sequest HT search engine. Analysis of MS spectra was performed using the following parameters: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.02 Da; (d) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; and (e) oxidation of methionine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node.

			Action	DRS
	210115masuta_again.pdResult	Other
	210115masuta_again.xlsx	Xlsx
	210115masuta_etp1.raw	Raw
	210115masuta_etp2.raw	Raw
	210115masuta_etp3.raw	Raw

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LFQ_TurboID-PSMA6_ETP_Tamavidin2-REV

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