Cells were rapidly washed three times with ice-cold PBS and lysed in 4% sodium deoxycholate in 100 mM Tris, pH 8.5 and heated at 95°C for 5min. The samples were tip-probe sonicated and centrifuged at 16,000 x g at 4°C for 15 min, protein quantified by BCA and normalized to 240 μg. Proteins were reduced with 10 mM tris(2-carboxyethyl)phosphine (TCEP) and alkylated with 40 mM 2-chloroacetamide (CAA) at 45°C for 5 min. Samples were digested with sequencing grade trypsin (Sigma #11418025001) and LysC (Wako, Japan) at 1:100 ratio of protease:protein at 37°C for 16h. Ten micrograms of peptide was removed for total proteome analysis and phosphopeptides enriched from the remainder of the sample using the EasyPhos protocol (Humphrey et al., 2018 (link)). For NFIX over-expression acquisitions, the sample preparation procedure was identical except only 20 μg of protein was digested and no phosphopeptide enrichment was performed.
Sequencing grade trypsin
Manufactured by Merck Group
Sourced in United States
Sequencing grade trypsin is a highly purified protease enzyme used in the preparation of protein samples for mass spectrometry analysis and protein sequencing. It is specifically designed to efficiently cleave peptide bonds, facilitating the generation of peptide fragments suitable for downstream identification and characterization.
Automatically generated - may contain errors
Lab products found in correlation
Paragon algorithm, by AB Sciex (4 mentions)
Ab4800 plus proteomics analyzer, by Thermo Fisher Scientific (4 mentions)
Calmix standards, by AB Sciex (4 mentions)
Dithiothreitol (dtt), by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Iodoacetamide, by Merck Group (3 mentions)
Nanoacquity uplc, by Waters Corporation (2 mentions)
Digital sonifier, by Emerson (2 mentions)
Synapt g2 hdms mass spectrometer, by Waters Corporation (2 mentions)
Hdac enzymes, by BPS Biosciences (2 mentions)
Spectramax m5 microtiter, by Molecular Devices (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lys c, by Fujifilm (1 mentions)
Sequencing grade lys c, by Fujifilm (1 mentions)
Pgex 4t 1, by Cytiva (1 mentions)
Taq dna polymerase, by New England Biolabs (1 mentions)
E coli top10, by Thermo Fisher Scientific (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Glutathione sepharose beads, by Cytiva (1 mentions)
37 protocols using sequencing grade trypsin
1
Quantitative Phosphoproteomics Sample Preparation
2
Protein Pretreatment and LC-MS/MS Analysis
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Protein pretreatment was performed for LC-MS/MS analysis. The protein samples were incubated in 10 mM DTT for 30 min at 56 °C. The samples were cooled at room temperature with additional iodacetamide (final concentration 20 mM) for carboxyamidomethylation. For in-solution digestion, sequencing grade trypsin (Sigma Aldrich, St. Louis, MO, USA) in 100 mM NH4HCO3 was added, and the samples were digested overnight at 37 °C. Nano High Resolution LC-MS/MS spectrometry (Thermo Fisher Scientific, Waltham, MA, USA) was performed using an electron and nano electron ionization spray with a quadruple and FT orbitrap mass analyzer. Peptide ions were detected in the full scan mode with a maximum at 2000 m/z. The maximum mass resolution was 140,000 at m/z 200. Proteins were identified by searching MS/MS spectra data against the Proteome Discoverer protein database.
3
Adipose Tissue Protein Extraction
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Adipose tissue homogenates were diluted 1:1 in 6 M guanidine in 100 mM Tris pH 7.5 containing 10 mM Tris(2-carboxyethyl)phosphine and 40 mM chloroacetamide, and heated at 95°C for 5 min. The lysate was tip-probe sonicated and centrifuged at 20,000 x g for 30 min at 4°C. The supernatant was precipitated with 6 volumes of acetone, overnight at −20°C. Pelleted protein were re-suspended in 10% trifluoroethanol in 100 mM Tris, pH 7.5 and quantified by BCA. Seven µg of protein was digested with 140 ng sequencing grade Lys-C (Wako) for 2 hr at 25°C followed by 140 ng of sequencing grade trypsin (Sigma Aldrich) overnight at 37°C. The digest was acidified to a final concentration of 1% trifluroacetic acid (TFA), and peptides purified using SDB-RPS solid-phase disks (Sigma Aldrich) and eluted with 1% ammonium hydroxide in 80% acetonitrile. Peptides were dried by vacuum centrifugation and resuspended in 0.1% TFA in 2% acetonitrile.
4
Purification and Characterization of Recombinant Protein
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Tris (hydroxymethyl) aminomethane (Tris), sodium dodecyl sulfate (SDS), Glycine, acrylamide (Acr), N, N′-methylenebisacrylamide (Bis), N, N, N′, N′-Tetramethylethylenediamine (TEMED) and ammonium peroxydisulfate (AP) were purchased from SERVA (Heidelberg, Germany). RC-DC™ Protein Assay Kit was from Bio-Rad (Hercules, CA, USA). Dithiothreitol (DTT), iodoacetamide (IAA), Triton X-100 and sequencing grade trypsin were from Sigma (St. Louis, MO, USA). Taq DNA polymerase, T4 ligase and all restriction enzymes used in cloning were from NEB (Ipswich, MA USA). E. coli Top10 was from Invitrogen (Paisley, UK). E. coli BL21-CodonPlus (DE3)-RIPL was from Stratagene (La Jolla, CA, USA). Expression vector pGEX-4T-1 and glutathione-sepharose beads were from Amersham Pharmacia Biotech (Uppsala, Sweden). Adult Sprague-Dawley rats (weighting 200–250 g) were purchased from the Center South University (Changsha, China). All the rats were allowed food and water ad libitum before being used in the experiments.
5
Protease Stability Assays for De Novo Proteins
6
Shotgun Sequencing of Venom Peptides
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In order to identify low molecular weight peptides that do not resolve well on 1D or 2D gels, shotgun sequencing was used. 3 µg of crude venom sample was dissolved in 50 µL of 100 mM ammonium carbonate to reduce and alkylate cysteine bonds with subsequent addition of 50 µL of 2% iodoethanol/0.5% triethylphosphine in acetonitrile. The sample was afterwards resuspended in 20 µL of 40 mM ammonium bicarbonate before overnight incubation (at 37 °C) with 750 ng of sequencing grade trypsin (Sigma-Aldrich). To stop digestion 1 µL of concentrated formic acid was added to each of the samples. Samples were lyophilised then resuspended in 20 µL of 5% ACN/0.5% FA, put into MS vials and subjected to LC–MS/MS analysis.
7
Proteomic Analysis of MDA-Modified HSA
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In vitro, MDA-modified HSA was analyzed for peptide modification. HSA was first alkylated with dithiothreitol (Sigma) and iodoacetamide (Sigma), precipitated, and thereafter digested by sequencing grade trypsin in ammonium bicarbonate and dimethyl sulfoxide. The peptides thus obtained were desalted on a C18 column and analyzed by liquid chromatography–mass spectrometry (MS).
Plasma proteins (10 μg) from each of 10 patients’ samples were dissolved in 50 mmol/l ammonium bicarbonate; reduced with 20 mmol/l DTT (Sigma) for 30 min at 56°C. iodoacetamide (66 mmol/l) in 50 mmol/l ammonium bicarbonate was added for alkylation at room temperature for 30 min. Sequencing-grade trypsin (1:3, trypsin: protein; Promega) was incubated with each sample (1:33 trypsin: protein) at 37°C, and formic (final concentration of 5%) added to stop this digestion. After 20 min, the samples were placed on a C18 Hypersep plate (Thermo Scientific), dried using a Speedvac, and re-suspended in 25 μl 2% Acetonitrile/0.1% formic acid.
Plasma proteins (10 μg) from each of 10 patients’ samples were dissolved in 50 mmol/l ammonium bicarbonate; reduced with 20 mmol/l DTT (Sigma) for 30 min at 56°C. iodoacetamide (66 mmol/l) in 50 mmol/l ammonium bicarbonate was added for alkylation at room temperature for 30 min. Sequencing-grade trypsin (1:3, trypsin: protein; Promega) was incubated with each sample (1:33 trypsin: protein) at 37°C, and formic (final concentration of 5%) added to stop this digestion. After 20 min, the samples were placed on a C18 Hypersep plate (Thermo Scientific), dried using a Speedvac, and re-suspended in 25 μl 2% Acetonitrile/0.1% formic acid.
8
Proteomics Analysis of Desulfovibrio Oralis
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Samples from the same three replicate D. oralis cultures used for the proinflammatory assay were also used for proteomic analysis, conducted as detailed in reference 100 (link). Cell pellets were resuspended in 4% sodium deoxycholate (SDC) in 100 mM ammonium bicarbonate (ABC) and ultrasonically disrupted. The crude protein extract was clarified by centrifugation and reduced with 10 mM dithiothreitol (DTT), and cysteine residues were blocked with 30 mM iodoacetamide (IAA). The proteins were then collected on top of a 10-kDa cutoff spin column filter. D. oralis spent culture medium samples (~12 ml) were concentrated on a 5-kDa filter. Collected proteins were washed with ABC and digested with sequencing-grade trypsin (Sigma). Peptides were collected by centrifugation, acidified to 1% formic acid (FA) followed by extraction with ethyl acetate, and concentrated. The peptide mixture was analyzed by an automated, two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) system (100 (link)) using the Ultimate 3000 RS system in-line with a Q Exactive Plus (QE+) mass spectrometer (Thermo, Fisher Scientific).
9
Protein Identification by Trypsin Digestion and MALDI-TOF MS
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2DE protein spots were excised and subjected to reduction (10 mM dithiothreitol), alkylation (50 mM iodoacetamide), and overnight in-gel digestion with sequencing grade trypsin (Sigma), in 50 mM ammonium bicarbonate at 37°C. Tryptic peptide digests were extracted in 50% acetonitrile containing 1% trifluoroacetic acid (TFA), and analyzed by MALDI-TOF-TOF MS using an AB4800-Plus Proteomics Analyzer (Applied Biosystems). To this end, tryptic digests were mixed with an equal volume of α-cyano-hydroxycinnamic acid saturated in 50% acetonitrile, 0.1% TFA, and 1 μL spotted onto an Opti-TOF 384-well plate, dried, and analyzed in positive reflector mode. TOF MS spectra were acquired using 500 shots at a laser intensity of 3000. TOF/TOF fragmentation spectra were acquired (500 shots at a laser intensity of 3900) for the ten most intense precursor ions. External calibration in each run was performed with CalMix standards (ABSciex) spotted onto the same plate. Fragmentation spectra were searched against the UniProt/SwissProt database (taxonomy: Serpentes) using the ProteinPilot v.4 and the Paragon algorithm (ABSciex) at ≥95% confidence, or manually interpreted and the deduced sequences BLASTed against the NCBI (http://blast.ncbi.nlm.nih.gov ) non-redundant database for protein class assignment by similarity.
10
Zooarchaeology by Mass Spectrometry
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We selected 12 unidentified bone samples (>1 cm in size) for zooarchaeology by mass spectrometry (ZooMS) analysis. We tested the protocol designed by Van Doorn et al. (2011 ) and used a warm (65 °C) ammonium bicarbonate buffer (50 mm ) to leach bone collagen without acid digestion. Then, trypsin digestion was carried out for 18 h at 37 °C using 0.5 μL of sequencing‐grade trypsin (Sigma). Enzymatic digestion was ended using 5 μL of 5% formic acid (FA), then the tryptic digests were purified and concentrated using C18 SpinTips (Thermo Scientific). Peptide elution was performed with 15 μL of 50% acetonitrile (ACN)/0.1% FA (v/v). Samples were dried overnight under a class 100 laminar flow hood. After re‐suspension, each sample (1 μL) was spotted on a target steel plate, and mixed with 1 μL α‐cyano‐4‐hydroxycinnamic acid (CHCA; Sigma) as matrix. The samples were then analysed in duplicate with MALDI‐ToF (Bruker) over a mass‐to‐charge range of 700–3500 m/z. Spectra were manually inspected and averaged using mMass (Strohalm et al., 2010 (link)), after setting a signal‐to‐noise ratio equal to 4. Taxonomic identification was performed comparing identified peptides with a database of peptide markers for all European, Pleistocene medium to large size mammals (Welker et al., 2016 (link)).
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