The role of Ptpn11E76K in MSC fate determination
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus
SUBMITTER: 
Kan Chen
PROVIDER: PXD040487 | iProX | Tue Feb 28 00:00:00 GMT 2023
REPOSITORIES: iProX
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Project description:Samples were subjected to LC–MS analysis using a dual pressure LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) connected to an electrospray ion source (Thermo Fisher Scientific) as recently described (PMID: 23017020). Peptide separation was carried out on an EASY nLC-1000 system (Thermo Fisher Scientific) equipped with a RP-HPLC column (75 μm × 30 cm) packed in-house with C18 resin (ReproSil-Pur C18–AQ, 1.9 μm resin, Dr. Maisch GmbH). A step-wise gradient from 95% solvent A (0.1% formic acid) and 5% solvent B (80% acetonitrile, 0.1% formic acid) to 50% solvent B over 60 min at a flow rate of 0.2 μl/min was used. Data acquisition mode was set to obtain one high resolution MS scan in the FT part of the mass spectrometer at a resolution of 240,000 full width at half-maximum (at m/z 400) followed by 20 MS/MS scans (TOP20) in the linear ion trap of the most intense ions using rapid scan speed. Unassigned and singly charged ions were excluded from analysis. Dynamic exclusion duration was set to 30 seconds.
2022-02-24 | PXD012490 | Pride
Project description:The expression profile during wound repair of cutaneous excisional wound (2x2 cm) was studied. The tissue was sampled from the centre of the open wound (day 3/7/14) or centre of the wound scar (day 21/35/70). The microarray slide were scanned in low intensity scan and high intensity scan mode.
2012-07-30 | GSE37947 | GEO
Project description:Ammonium hydroxide and pyrrolidine eluents were dried (SpeedVac) and reconstituted in 25 ul sample loading buffer (0.1% TFA/2% acetonitrile). Eluent from phosphotyrosine immunoprecipitation was dried and reconstituted in 35 ul of the loading buffer. An LTQ Orbitrap XL (ThermoFisher) in-line with a Paradigm MS2 HPLC (Michrom bioresources) was employed for acquiring high-resolution MS and MS/MS data. Ten microliters of the phospho-enriched peptides were loaded onto a sample trap (Captrap, Bruker-Michrom) in-line with a nano-capillary column (Picofrit, 75 um i.d.x 15 um tip, New Objective) packed in-house with 10 cm of MAGIC AQ C18 reverse phase material (Michrom Bioresource). Two different gradient programs, one each for MOAC and phosphotyrosine immunoprecipitation samples, were used for peptide elution. For MOAC samples, a gradient of 5-40% buffer B (95% acetonitrile/1% acetic acid) in 135 min and 5 min wash with 100% buffer B followed by 30 min of re-equilibration with buffer A (2% acetonitrile/1% acetic acid) was used. For phosphotyrosine immunoprecipitation samples, which were a much less complex mixture of peptides, 5-40% gradient with buffer B was achieved in 75 min followed by 5 min wash with buffer B and 30 min re-equilibration. Flow rate was ~0.3 ul/min. Peptides were directly introduced into the mass spectrometer using a nano-spray source. Orbitrap was set to collect 1 MS scan between 400-2000 m/z (resolution of 30,000 @ 400 m/z) in orbitrap followed by data dependent CID spectra on top 9 ions in LTQ (normalized collision energy ~35%). Dynamic exclusion was set to 2 MS/MS acquisitions followed by exclusion of the same precursor ion for 2 min. Maximum ion injection times were set to 300 ms for MS and 100 ms for MS/MS. Automatic Gain Control (AGC) was set to 1xe6 for MS and 5000 for MS/MS. Charge state screening was enabled to discard +1 and unassigned charge states. Technical duplicate data for each of the MOAC elutions (ammonium hydroxide and pyrrolidine) and triplicate data for the phosphotyrosine immunoprecipitation samples were acquired. RAW files were converted to mzXML using msconvert and searched against the Swissprot Human protein database (2013-Jan release) appended with common proteomics contaminants and reverse sequences as decoys. Searches were performed with X!Tandem (version 2010.10.01.1) using the k-score plugin. For all searches the following search parameters were used: Parent monoisotopic mass error of 50 ppm; fragment ion error of 0.8 Daltons; allowing for up to 2 missed tryptic cleavages. Variable modifications were oxidation of Methionine (+15.9949@M), carbamidomethylation of Cysteine (+57.0214@C), and phosphorylation of Serine, Threonine, and Tyrosine (+79.9663@[STY]). The search results were then post-processed using PeptideProphet and ProteinProphet.
2014-01-09 | PXD000658 | Pride
Project description:The mass spectrometry data were acquired using a Q Exactive HF mass spectrometer coupled with an UltiMate 3000 RSLCnano liquid chromatography system. Peptide samples were dissolved in a loading buffer, introduced via an autosampler, and separated on an analytical column (75 μm × 25 cm, C18, 1.9 μm, 120 Å). An analytical gradient was established using two mobile phases (mobile phase A: 0.1% formic acid, 3% DMSO; mobile phase B: 0.1% formic acid, 3% DMSO, 80% ACN). The flow rate of the liquid phase was set to 300 nL/min. Data were acquired in DDA mode, with each scan cycle including one MS full scan (R = 60 K, AGC = 3e6, max IT = 25 ms, scan range = 350–1500 m/z), followed by 20 MS/MS scans (R = 15 K, AGC = 1e5, max IT = 50 ms). The HCD collision energy was set to 27. The quadrupole isolation window was set to 1.4 Da, and the dynamic exclusion time for ion repeat acquisition was set to 24 s.
2024-09-10 | PXD055763 |
Project description:The expression profile during wound repair of cutaneous excisional wound (2x2 cm) was studied. The tissue was sampled from the centre of the open wound (day 3/7/14) or centre of the wound scar (day 21/35/70). The microarray slide were scanned in low intensity scan and high intensity scan mode. Wound tissue (day 3, day 7, day 14, day 21, day 35, day 70, n = 4 per interval) vs. control - uninjured skin (day 0, n=4 ). In each interval two biological replicates of four were labeled with flip dyes.
2012-07-29 | E-GEOD-37947 | biostudies-arrayexpress
Project description:In this study we analyzed and compared total proteome and secretome of the wild-type and JN1 mutant strains of Bordetella pertussis. The single-nucleotide transversion in the 5’-UTR of the rplN gene of JN1 mutant led to the increased transcription of the whole operon encoding ribosomal proteins and of the adjoining rpoA gene. These events led to the downregulation and decreased secretion of virulence factors on the background ofgenerally deregulated expression of B. pertussis genome. To get deeper inside in the molecular mechanisms of the observed genome deregulation we then performed the immunoprecipitation of RpoA and compared its binding partners in wild-type and JN1 mutant strains. Nano Reversed phase column (EASY-Spray column, 50 cm x 75 µm ID, PepMap C18, 2µm particles, 100Å pore size) was used for LC/MS analysis. Mobile phase buffer A was composed of water and 0.1% formic acid. Mobile phase B was composed of acetonitrile and 0.1% formic acid. Samples were loaded onto the trap column (Acclaim PepMap300, C18, 5µm, 300Å wide Pore, 300 µm x 5 mm) at a flow rate of 15 μl/min. Loading buffer was composed of water, 2% acetonitrile and 0.1% trifluoroacetic acid. Peptides were eluted with gradient of B from 4% to 35% over 60 min at a flow rate of 300 nl/min. Eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a Thermo Orbitrap Fusion (Q-OT- qIT, Thermo). Survey scans of peptide precursors from 350 to 1400 m/z were performed at 120K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS was performed by isolation at 1.5 Th with the quadrupole, HCD fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion trap. The MS 2 ion count target was set to 104 and the max injection time was 35 ms. Only those precursors with the charge state 2–6 were sampled for MS 2. The dynamic exclusion duration was set to 45 s with a 10ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 2s cycles (76). The data were analyzed and quantified with label-free quantification (LFQ) algorithms in MaxQuant v1.6.3.3 (77) and the Andromeda search engine(78). The false discovery rate (FDR) parameter was set to 1 % for both proteins and peptides. The enzyme specificity of trypsin was set as C-terminal to Arg and Lys. Carbamidomethylation was set as the fixed modification, while N-terminal protein acetylation and methionine oxidation were variable modifications. Maximal number of missed cleavages was set to 2. All hits identified in searches as contaminants were filtered out. The data were searched against Bordetella pertussis reference proteome database (strain Tohama I / ATCC BAA-589 / NCTC 13251).
2020-11-09 | PXD020023 | Pride
Project description:Four bile samples collected from patients with a malignant (PAC, CC) or a nonmalignant (CP, BS) biliary stenosis were used for comparative proteomic analysis. Briefly, bile samples were centrifuged at 16,000/g/ for 10 min at 4 C. Each supernatant was delipided with Cleanascite (Biotech Support Group, North Brunswick, NJ, USA) and ultrafiltrated using a 3 kDa filter cut-off (YM-3 centricon, Millipore, Bedford, MA, USA). For each sample, 50 ug of proteins were mixed with 0.5 M triethylammonium bicarbonate (TEAB) buffer pH 8.0 to a total volume of 100 uL. One ug of bovine lactoglobulin (LACB) was spiked in each sample as an internal standard. Proteins were reduced and alkylated before digestion of N-linked oligosaccharides from glycoproteins using PNGase F (Sigma-Aldrich, St. Louis, MO, USA). Proteins were then digested with trypsin and the resulting peptides were tagged with one of the isobaric tags from the iTRAQ reagents 4plex kit (AB Sciex, Foster City, CA, USA) according to the manufacturer's instructions. The four samples were then pooled and dried under vacuum. Labeled peptides were fractionated according to their isoelectric point on an Agilent 3100 OFFGEL fractionator using 24 cm pH 3-10 linear immobilized pH gradients (GE Healthcare, Chalfont St. Giles, UK) following manufacturer's instructions. Fractions were then recovered in separate tubes for high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). MS analysis was performed on a LTQ Orbitrap XL from Thermo Electron (San Jose, CA, USA) equipped with a NanoAcquity HPLC system from Waters. Peptides were trapped on a home-made 5 um 200 A Magic C18 AQ (Michrom, Auburn, CA, USA) 0.1 x 20 mm pre-column and separated on a home-made 5 um 100 A Magic C18 AQ (Michrom) 0.75 x 150 mm column with a gravity-pulled emitter. The analytical separation was run for 65 min using a gradient of H_2 O/formic acid (FA) 99.9%/0.1% (solvent A) and CH_3 CN/FA 99.9%/0.1% (solvent B). The gradient was run at a flow rate of 220 nL/min as follows: 5% B for 1 min, from 5 to 35% B in 54 min, from 35 to 80% B in 10 min. For MS survey scans, the OT resolution was set to 60,000 and the ion population was set to 5 x 10^5 with an m/z window from 400 to 2000. Maximum of 3 precursors were selected for both collision-induced dissociation (CID) in the LTQ and high-energy C-trap dissociation (HCD) with analysis in the Orbitrap (OT). For MS/MS in the LTQ, the ion population was set to 1 x 10^4 (isolation width of 2 m/z) while for MS/MS detection in the OT, it was set to 2 x 10^5 , with resolution of 7,500, first mass at m/z = 100, and maximum injection time of 750 ms. The normalized collision energies were set to 35% for CID and 60% for HCD. Peak lists from MS analysis were searched against Uniprot_Swissprot database (version 2011_2) using Phenyx protein identification software (GeneBio, Geneva, Switzerland). /Homo Sapiens/ taxonomy was specified for database searching. The parent ion tolerance was set to 10 ppm. Variable amino acid modifications were oxidized methionine and deamidated asparagine. Carbamidomethylation of cysteines and iTRAQ-labeled peptides on the amino terminus and lysine were set as fixed modifications. Trypsin was selected as the enzyme, with one potential missed cleavage, and the normal cleavage mode was used. Only one search round was used with selection of "turbo" scoring. The peptide p value was 10^-2 for LTQ-OT data. Protein and peptide scores were set up to maintain the false positive rate below 5%.
2013-06-20 | PXD000284 | Pride
Project description:Tags 126, 127N, and 128C were assigned to control replicates, 127C, 128N, and 130C were assigned to 0.5 mM SA-treated replicates, and 129C, 130N, and 131 were assigned to 1.0 mM SA-treated replicates. Labeled samples were mixed together and fractionated by high-pH reverse phase HPLC. Twelve concatenated fractions were analyzed. Peptides from each fraction were separated using a gradient of 3 to 27% of 90% acetonitrile\0.1% formic acid over 180 minutes and were electrosprayed at 2.6 kV into an Orbitrap Fusion mass spectrometer operating in data-dependent mode with positive polarity. Quadrupole isolation was enabled, and survey scans were recorded in the Orbitrap at 120,000 resolution over a mass range of 400-1500 m/z. The automatic gain control (AGC) target was set to 1,600,000 and the maximum injection time was set to 50 milliseconds (msec). The most abundant precursor ions (intensity threshold 5,000) were fragmented by collision-induced dissociation (35% energy) and fragment ions were detected in the linear ion trap (AGC 32,500, 50 msec maximum injection). Analyzed precursors were dynamically excluded for 150 seconds. Multiple MS2 fragment ions were captured using isolation waveforms with multiple frequency notches and fragmented by high energy collision-induced dissociation (65% normalized collision energy). MS3 spectra were acquired in the Orbitrap (AGC 250,000; maximum injection time 200 msec, 50,000 resolution scanning from 100-1000 m/z).
2021-07-19 | MSV000087853 | MassIVE
Project description:To achieve deep coverage of M. smegmatis proteome, 240 μg proteins were reduced with 5 mM of dithiothreitol (DTT), alkylated with 20 mM of iodoacetamide (IAA). The alkylated proteins were separated by a 10% SDS-PAGE for 8 cm, and stained with Coomassie Blue G250. Gel lane was excised into 13 fractions based on the molecular wight (MW) and protein abundance, and digested with and Ac-LysargiNase (Zhang et al., 2019) (12.5 ng/μL) at 37 °C for 12-24 h. The extracted peptides were desalted with a homemade C18 StageTip (Zhai et al., 2013), dried and dissolved in loading buffer (1% acetonitrile, ACN and 1% formic acid, FA) for MS analysis. The dissolved peptides (500 ng) were analyzed as described previously. Briefly, the liquid chromatography tandem mass spectrometry (LC-MS/MS) consisted of an EASY-nLC 1200 system (Thermo Fisher Scientific, San Jose, CA, United States) equipped with a self-packed capillary column (75 μm i.d. × 15 cm, 3 μm C18 reversed-phase fused silica) coupled to an Orbitrap Fusion Lumos (Thermo Fisher Scientific). For full MS scans, the automatic gain control (AGC) was 5.0 × 105, the scan ranged from 300 to 1,400 m/z at a resolution of 1.2 × 105 and a maximum injection time (MIT) of 50 ms. For the MS2 scan, only spectra with a charge state of 2-6 were selected for fragmentation by higher energy collision-induced dissociation (HCD) with a normalized collision energy of 32%, AGC of 1 × 105, and MIT of 35 ms. The dynamic exclusion was set to 30 s.
2022-09-29 | PXD037080 |
Project description:To achieve deep coverage of M. smegmatis proteome, 240 μg proteins were reduced with 5 mM of dithiothreitol (DTT), alkylated with 20 mM of iodoacetamide (IAA). The alkylated proteins were separated by a 10% SDS-PAGE for 8 cm, and stained with Coomassie Blue G250. Gel lane was excised into 13 fractions based on the molecular wight (MW) and protein abundance, and digested with Ac-Trypsin (Wu et al., 2016) (12.5 ng/μL) at 37 °C for 12-24 h. The extracted peptides were desalted with a homemade C18 StageTip (Zhai et al., 2013), dried and dissolved in loading buffer (1% acetonitrile, ACN and 1% formic acid, FA) for MS analysis.The dissolved peptides (500 ng) were analyzed as described previously. Briefly, the liquid chromatography tandem mass spectrometry (LC-MS/MS) consisted of an EASY-nLC 1200 system (Thermo Fisher Scientific, San Jose, CA, United States) equipped with a self-packed capillary column (75 μm i.d. × 15 cm, 3 μm C18 reversed-phase fused silica) coupled to an Orbitrap Fusion Lumos (Thermo Fisher Scientific). For full MS scans, the automatic gain control (AGC) was 5.0 × 105, the scan ranged from 300 to 1,400 m/z at a resolution of 1.2 × 105 and a maximum injection time (MIT) of 50 ms. For the MS2 scan, only spectra with a charge state of 2-6 were selected for fragmentation by higher energy collision-induced dissociation (HCD) with a normalized collision energy of 32%, AGC of 1 × 105, and MIT of 35 ms. The dynamic exclusion was set to 30 s.
2022-09-29 | PXD037081 |