Project description:Proteomic analysis of pervanadate-induced tyrosine-phosphorylated proteins in hepatocellular carcinoma WRL 68 cells Protein tyrosine phosphorylation plays critical roles in modulating biological processes such as cellular proliferation, differentiation, migration, apoptosis and metabolism. To profile tyrosine phosphorylated (pTyr) proteins as well as search novel pTyr proteins as cross-talk points among different cellular pathways, we developed a rapid and efficient approach to identify cellular pTyr proteins and their complexes by a combination of subcellular proteomics approach with signal transduction strategies. Human hepatocytic cells from WRL68 cell line were treated with pervanadate (POV), subfractionated into four fractions and then subjected to immunoaffinity purification with anti-pTyr antibody. The eluted mixtures of the anti-pTyr purification were identified by LC-MS/MS. Subcellular fractionation and affinity purification of tyrosine-phosphorylated proteins: WRL68 cells were first grown to 80% confluence in MEM complete medium and then the medium replaced with serum free media. After 15 h, the cells were either untreated or stimulated with 0.1 mM pervanadate (1 mM sodium orthovanadate, 3 mM H2O2) for 10 min. 150-mm cultures of WRL 68 cells were rinsed twice with 4? PBS and then scraped from the dish in 750?l of hypotonic buffer (10 mM Tris, 1 mM NaF, 10 mM IAA, pH 7.5) containing a cocktail of protein inhibitors. After a 20-min incubation on ice, the cells were passed about five times through a 25-g needle. The resulting lysate was subjected to a 15min centrifugation at 1000 rpm at 4?, after which the pellet was resuspended in 250?l of hypotonic buffer and re-extracted by a second round of the trituration and centrifugation. The supernatants of the first and second spins were combined, adjusted to 0.25 M NaCl, and separated into cytosolic (supernatant) and membrane (pellet) fractions bycentrifugation at 19,000 rpm (43,000 g) for 90 min at 4?. All the pellets and total cell lysate were resuspended in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid sodium) containing 1mM pervanadate with a cocktail of protein inhibitors with sonication aid. Cleared cell lysates were incubated overnight at 4? with 30?l monoclonal anti-phosphotyrosine-agrose (Sigma). Precipitated immune complexes were washed three times with 1×HNTG (20 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, 10% Glycerol, pH 7.5) and then eluted with 100 mM phenyl phosphate (Sigma) in lysis buffer at 4?. Enzyme digestion, mass spectrometry and protein identification: The sample was digested according to the published method. Chromatography was performed using a surveyor LC system (Thermo Finnigan,SanJose,CA) on C18 reverse phase column(RP, 180 µm x 150 mm, BioBasic® C18, 5 µm, Thermo Hypersil-Keystone). The pump flow rate is split 1:100 for a colum flow rate of 1.5 µL/min.The column effluent is directly electrosprayed using the orthogonal metal needle source without further splitting.Mobile phase A is 0.1% formic acid in water,and the B mobile phase is 0.1% formic acid in acetonitrile. The separation of peptides obtained by enzymatic digest of bile sample was achieved with a gradient of 2-80% B over 360 min.The column effluent from the reverse phase column was analyzed by LCQ Deca XP ion-trap mass spectrometer.The micro-electrospray interface uses a 30 µm metal needle that is orthogonal to the inlet of the LCQ.The mass spectrometer was set that one full MS scan was followed by three MS/MS scans on the three most intense ion from the MS spectrum with the following Dynamic Exclusion™ settings: repeat count, 1; repeat duration, 0.5 min; exclusion duration, 3.0 min. The acquired MS/MS spectra were automatically searched against protein database for human proteins (SWISS-PROT/TrEMBL) using the TurboSEQUEST program in the BioWorks™ 3.0 software suite. An accepted SEQUEST result had to have a ?Cn score of at least 0.1 (regardless of charge state) and Xcorr (one charge?1.5, two charges?2.0, three charges?2.5). Single peptides that alone identify a protein were manually validated after meeting the above criteria. Bioinformatics analysis: The pI and MW of the proteins were analyzed using ExPASy proteomics tools accessed from http://cn.expasy.org/tools/#proteome. The grand average hydropathicity (GRAVY) values were determined according to Kyte-Doolittle. The protein subcellular location annotation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/). The protein function family was categorized according to Gene Ontology (GO) annotation terms extracted by InterPro (http://www.ebi.ac.uk./interpro/). The annotation of protein phosphorylation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/) and PhosphoSite (http://www.phosphosite.org). The kinases were annotated according to human kinome. Keywords: other

Project description:Sample preparation was conducted using an adaptation of our SimPLIT workflow10. Four cell pellets corresponding to four different colorectal cancer cell lines were lysed in a buffer containing 1% sodium deoxycholate (SDC, Merck, Cat. No. 30970), 100 mM triethylammonium bicarbonate (TEAB, Merck, Cat. No. T7408), 10% isopropanol and 50 mM NaCl, freshly supplemented with 5 mM TCEP (Thermo, Bond-breaker Cat. No 77720), 10 mM iodoacetamide (IAA, Merck, Cat. No. I1149), universal nuclease 1:2000 vol/vol (Pierce, Cat. No. 88700) and Halt protease and phosphatase inhibitor cocktail (Thermo, Cat. No. 78442, 100X) with 5 min of bath sonication. Protein concentration was measured with the Quick Start Bradford protein assay (Bio-Rad, Cat. No. 5000205). Aliquots of 30 μg of total protein from each cell line were transferred in a 96-well plate according to the 58-plex design. An E. coli protein extract (Bio-Rad, Cat. No. 1632110) was added at 1 μg or 0.5 μg across the human cell lysates. The plate was dried and the samples for the arginine-specific cleavage set (TrypR) were reconstituted in 25 μL (TMT11 subset) or 12.5 μL (TMT18 subset) of 100 mM TEAB buffer followed by the addition of 10 μL TMT11 (stock 20 μg/μL, Thermo Fischer Scientific, Cat. No. A34808) or 5 μL TMTpro18 (stock 25 μg/μL, Thermo Fischer Scientific, Cat. No. A52045), and 1h reaction at room temperature for protein-level TMT labelling. The TMT labelled samples were SpeedVac dried, and all samples (labelled and unlabelled) were reconstituted in 20 μL of 100 mM TEAB buffer. Proteins were digested overnight at room temperature by the addition of 2 μL LysC (Thermo Fischer Scientific, Cat. No. 90307) or Trypsin (Thermo Fischer Scientific, Cat. No. 90059) stock solution of 500 ng/μL in 0.1% formic acid (final enzyme 50 ng/μL, 1:30) for the LysC and TrypR sets respectively. The peptides were then fully labelled by another addition of 10 μL TMT11 or 5 μL TMTpro18. The reaction was quenched after 1h with the addition of 2 μL 5% hydroxylamine solution and premix samples were prepared by pooling 0.5 μL from each sample into 100 μL of 0.1% TFA for each sub-plex separately. In each premix, SDC was precipitated with addition of formic acid at 2% and centrifugation at 20,000 × g for 5 minutes. Supernatants were transferred into clean vials for LC-MS analysis. Following evaluation of the premix runs, samples were pooled into four sub-plexes, diluted with 100 μL of HPLC water, heated at 95 °C for 5 min, cooled down and heated for another 5 minutes. Lastly, each pool was acidified with formic acid at 2%, the precipitated SDC was removed by centrifugation and supernatants were SpeedVac dried.High-pH Reversed-phase Peptide FractionationAll four sub-plexes were pooled in 100 μL of 0.1% (v/v) ammonium hydroxide and the final peptide pool was fractionated with high pH Reversed-Phase chromatography using the XBridge C18 column (2.1 × 150 mm, 3.5 μm, Waters) on an UltiMate 3000 HPLC system over a 1% gradient in 35 min. Mobile phase A was 0.1% (v/v) ammonium hydroxide and mobile phase B was 0.1% ammonium hydroxide (v/v) in acetonitrile. Fractions were collected every 30 sec and eventually pooled into 45 samples for LC-MS analysis.

Project description:Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.

Project description:Bacteria was grown at 30C in 3 different conditions, i.e. SYN (syngas and minimal medium- ATCC no 1789), AC (0.3% acetate in minimal medium- ATCC no 1789) and TSB (tryptic soy broth). After harvesting by centrifugation, O. carboxidovorans pellets (1g) were lysed in 100 mM Tris-Cl pH 8.0, 2% Triton X-100, 2.6 mg/ml sodium azide, 8 mM PMSF by sonication on ice (4 pulses of 15 s duration each). For each condition of growth 4 samples (1g pellets) were separately treated (i.e. lysed and processed further). The supernatants were treated with 50% cold TCA, and the precipitated protein washed with acetone. The pellets were resuspended in solubilization buffer (7M urea, 20 mM tris-Cl, pH 8.0, 5 mM EDTA, 5 mM MgCl2, 4% CHAPS), and protein concentration was determined using the Plus One 2-D Quant Kit (Amersham) following the manufacturers instructions. Protein samples from each treatment were stored at -80 C. One hundred micrograms of each protein sample was resuspended in 0.1 M ammonium bicarbonate, 5% HPLC grade ACN, reduced in 5 mM DTT (65 C, 5 min), alkylated in 10 mM iodoacetamide (30 C, 30 min), and then trypsin digested until there was no visible pellet (1:50 w/w 37 C, 16 h). Peptides were desalted using a peptide microtrap (Michrom BioResources, Auburn, CA) and eluted using a 0.1% TFA, 95% ACN solution. Desalted peptides were dried in a vacuum centrifuge and resuspended in 20 ?l of 0.1% formic acid. Peptides were separated by strong cation exchange (SCX) liquid chromatography (LC) followed by reverse phase (RP) LC coupled directly in line with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). 2DLC ESI MS/MS was done exactly as described (1). All searches were done using TurboSEQUEST (Bioworks Browser 3.2; Thermo Electron). Mass spectra and tandem mass spectra were searched against all annotated proteins from the strain OM5 including all the annotated plasmid-encoded proteins. Cysteine carbamidomethylation and methionine oxidation (single and double) were included in the search strategy. We used the reverse database functionality in Bioworks 3.2 and searched MS2 data against a reversed OM5 database using identical search criteria.

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:This dataset contains the raw data from the JASMS paper with the same title and DOI: 10.1007/s13361-012-0544-2. We compared the results of shotgun proteomic experiments of a trypsin digest of SUM52 cell lysate using 2D-LC fractionated (strong cation exchange and reversed phase of 6 SCX fractions) with FAIMS based fractionation. The FAIMS fractionation was performed using two different methods, an internal stepping method where the compensation voltage (CV) is cycled through 6 different voltages during the LC run (the three most abundant ions are fragmented at each CV. To match with the other methods this was run in six replicates. The other method was termed external stepping, for this method individual LC runs were performed at the six CV’s used earlier (-25 V to -50 V in 5 V steps). The three most abundant ions were again fragmented. For all three of these methods data was collect using electron transfer dissociation (ETD) and collision induced dissociation (CID) giving a total of 6 datasets with 6 raw files in each. SUM52 breast cancer carcinoma cells were cultured in HPMi-1640 formulation, supplemented with 2 mM L-glutamine, 1% Pen-Strep and 10% PBS at 37 oC in a 5% CO2 atmosphere. Lysis buffer contained Triton X-100 (0.5%), NaCl (0.15 M), PhosphoStop phosphatase inhibitor tablet (Roche) and Mini-complete protease inhibitor (Roche). The lysed cells were digested with trypsin at a ratio of 50:1 protein to enzyme. Part of the digest was fractionated with strong cation exchange using a Polysulfoethyl A column. All fractions and crude digest were desalted prior to mass spectrometry analysis. Approximately 1.66 µg of digest was loaded per LC-MS/MS run onto a 150mm Acclaim PepMap100 C18 column and separated over a 30 minute gradient from 3.2% acetonitrile (0.1% formic acid) to 44% acetonitrile (0.1% formic acid). The raw files were searched using proteome discoverer 1.2 against the human IPI database V3.81 supplemented with known contaminants and the reverse sequences. The files were searched with both sequest and mascot (v2.2.0). The MSF files were manually filtered using mascot significance scores and XCORR vs change state to give a 1% FDR, for filter setting see the linked paper. Included in the downloadable files is a xls file which documents the raw files listed and the names they had for the original searches, the names have been changed for clarity of the dataset.

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CID and EAD MS/MS library of steroids

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