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Pierce trypsin protease

Manufactured by Thermo Fisher Scientific
Sourced in United States

Pierce trypsin protease is a proteolytic enzyme used for the digestion and cleavage of proteins. It functions by catalyzing the hydrolysis of peptide bonds, primarily at the carboxyl side of lysine and arginine residues.

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20 protocols using pierce trypsin protease

1

Sorafenib Mass Spectrometry Analysis

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Methanol (≥99.9%), acetonitrile (ACN) and deionized water, as well as LC-MS CHROMASOLV, were purchased from Honeywell (Wunstorfer, Strasse, Seelze, Germany). Trifluoroacetic acid (TFA) and formic acid (FA) were purchased from Fisher Scientific (Loughborough, UK). Hydrochloric acid (HCl) (37%) was purchased from VWR chemicals (France). C18 columns, lysis buffer, Pierce trypsin protease, lysyl-endopeptidase LysC and Pierce protease inhibitor tablets were obtained from Thermo Scientific (Rockford, IL, USA). Bradford’s reagent and bovine serum albumin were bought from Sigma-Aldrich (St. Louis, MO, USA). Sorafenib was obtained from BioVision (Milpitas, CA, USA #BAY 43-9006).
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2

Purified Protein Digestion and Desalting

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Purified proteins were digested, first with 20 μg lysyl endopeptidase (Lys-C) (Wako) for 3 h at 20 °C, diluted in 50 mM Tris pH 8, and then with 20 μg of trypsin (Pierce trypsin protease, mass spectrometry (MS) grade, Thermo Fisher Scientific) overnight at 20 °C. A mixture of peptides was then acidified with 0.5% (final concentration) of formic acid (FA) and subsequently desalted using a SEP-PakVac tC18 cartridge column (Waters). C18 cartridges were conditioned with 0.5 ml of 100% acetonitrile (ACN), followed by 0.5 ml of 50% ACN and 0.1% FA, and finally ∼1.2 ml of 0.1% trifluoroacetic acid (TFA). Digested samples were loaded onto the conditioned C18 cartridge column, washed twice with 1 ml of 0.1% TFA, and eluted with 600 μl of 50% ACN and 0.1% FA. Eluates were lyophilized at −80 °C using Heto Drywinner (Thermo Fisher Scientific).
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3

Proteomics Analysis of H. pylori

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H. pylori cell pellets were lysed and its protein was extracted using Norgen's Proteospin™ total protein purification kit (Norgen Biotek Corporation, Canada). Cells were resuspended in 50 μl lysis buffer and centrifuged at 14,000 × g for 2 min. The supernatant was transferred into a filter column fitted in an elution tube, and centrifuged at 14,000 × g for 1 min. One microliter of protease inhibitor (Halt Protease and Phosphatase Inhibitor; Thermo Fisher Scientific) was added to the tube. Protein concentrations were determined by Bradford assay (Bio-Rad, USA). Each tube of protein sample was reduced with a volume of 10 mM dithiothreitol (DTT; Bio-Rad), and alkylated in the dark with a volume of 20 mM iodoacetamide (IAA; Bio-Rad). The samples were incubated at room temperature for 30 min and added with Pierce™ trypsin protease (Thermo Scientific) to digest the proteins at 1:50 trypsin:protein. The samples were then incubated at 4°C for 30 min, and subsequently at 37°C for overnight. The extracted protein was then treated for liquid chromatography mass spectrophotometry according to a previous study (Chan et al., 2015 (link)).
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4

Protein Extraction and Digestion Protocol

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Cells were lysed using the lysis buffer consisting of 3% sodium deoxycholate, 6 M urea, and 1 × Halt™ protease and phosphatase inhibitor cocktail (Thermo Scientific, VIC, Australia) in 50 mM ammonium bicarbonate (AMBIC) buffer. After sonicating and agitating, samples were centrifuged (14,000×g for 15 min at 4 °C) and the supernatant was collected for BCA assay. Protein samples were reduced with 15 mM dithiothreitol at 60 °C for 30 min and then alkylated with 20 mM iodoacetamide at room temperature for 30 min in the dark. These samples were diluted with 50 mM AMBIC buffer six times before digestion which was performed with the Pierce™ Trypsin Protease (Thermo Scientific) at a trypsin:protein ratio of 1:50 (w/w) at 37 °C overnight. On the second day, formic acid (FA) was added to stop digestion, and the precipitates were removed by centrifugation. The supernatants were desalted and cleaned using the Pierce™ C18 Spin Tips (Thermo Scientific). Collected samples were dried using the SpeedVac centrifugal evaporator and then resolved in 0.1% FA to a final concentration of 1 μg/μL.
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5

Zooarchaeological Bone Analysis by ZooMS

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Highly fragmented faunal bones were analysed by ZooMS to confirm taxonomic identifications (Supplementary Data 7). Samples were analysed following established protocols86 in which bone samples were demineralized in 0.6 M hydrochloric acid (HCI) for 18 h. The HCl was removed and the sample was rinsed three times in pH 8 solution of 50 mM ammonium bicarbonate (AmBic). After rinsing, the sample was incubated at 70 °C for an hour in 100 µL of 50 mM AmBic. Fifty microlitres of the resulting supernatant was treated with trypsin (Pierce™ Trypsin Protease, Thermo Scientific) at 37 °C for 18 h. Following digestion, the samples were subjected to C18 cleanup (Pierce™ C18 Tips, Thermo Scientific), mixed with a matrix solution of α-cyano-4-hydroxycinnamic of 10 mg/mL in 50% ACN/0.1% trifluoroacetic acid (TFA) and allowed to co-crystallize. Analysis was carried out on an Autoflex MALDI-TOF Bruker Ultraflex II (Bruker Daltonics, Bremen). The resulting mass spectra were screened for diagnostic markers using the FlexAnalysis software and compared against a reference library87 (link)–89 (link) and analysed using mMass. Samples were analysed alongside multiple blanks which all returned negative results and were determined to be empty.
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6

Demineralization and Proteomic Analysis of Bone

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Bone samples were first demineralized in 0.6 M hydrochloric acid (HCl) for at least 18 h, after which the HCl supernatant containing the acid-insoluble fraction was removed and kept aside for the acid-soluble protocol, see below [96 (link),99 (link)]. The demineralized bone was rinsed thrice with 50 mM ammonium bicarbonate (AmBic), incubated at 70°C in 100 µl of 50 mM AmBic, and 50 µl of the resulting supernatant was treated with 0.1 µg trypsin (Pierce™ Trypsin Protease, Thermo Scientific) at 37°C for 18 h.
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7

Collagen Extraction for Zooarchaeological Analyses

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For the 22 samples that bone collagen was extracted for stable isotope analysis, ZooMS was carried out using the resulting lyophilized collagen from the stable isotope preparation protocol [101 (link),102 (link)]. In brief, samples were demineralized in 0.5 M HCl for 1–5 days, rinsed in Milli Q water, treated with pH 3 water and demineralized at 70°C for 24 h. The resulting supernatant was filtered using Ezee Filters and freeze dried in order to lyophilize the collagen. Less than 0.1 µg of the lyophillized collagen was then eluted in 50 mM AmBic and treated with 0.1 µg trypsin (Pierce™ Trypsin Protease, Thermo Scientific) and incubated at 37°C for 18 h.
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8

Trypsin-Digested OleH Analyzed by MALDI-TOF MS

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Mass spectrometry (MS)-grade Pierce trypsin protease (Thermo Scientific, Rockford, USA) was dissolved in 50 mM acetic acid at a final concentration of 10 ng/μl. A digestion reaction was carried out for 16 h at 37°C and consisted of 3 μl of trypsin solution, 10 μl of OleH, followed by addition of 10 μl 0.5 mM ammonium bicarbonate. MS was performed on the digested OleH with an Axima TOF2 MALDI-TOF MS (Shimadzu Biotech, Manchester, UK). A 0.5-μl aliquot of matrix solution (alpha-cyano-4-hydroxycinnamic acid, 10 mg/ml in 50% acetonitrile-0.1% [vol/vol] trifluoroacetic acid) was deposited onto the target and left for 5 s before being removed. The residual solution was allowed to air dry, and 0.5 μl of the sample solution was deposited onto the precoated sample spot. A 0.5-μl aliquot of matrix solution was added to the deposited sample and allowed to air dry. The sample was subsequently analyzed in positive-ion reflectron mode. Protein identification was carried out via peptide mass fingerprinting (PMF) using the Mascot search engine (http://www.matrix-science.com). The monoisotopic positive-ion data ± 0.25 Da were searched using the following parameters: NCBInr database or Swiss-Prot, taxonomy all entries, and trypsin digest with one missed cleavage.
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9

Quantitative Proteomic Sample Preparation

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Acetonitrile (ACN), methanol (≥99.9%), chloroform, LC-MS CHROMASOLV, and deionized water were bought from Honeywell (Wunstorfer, Strasse, Seelze, Germany). Hydrochloric acid (HCl) (37%) was bought from VWR Chemicals (France). Formic acid (FA) and trifluoroacetic acid (TFA) were purchased from Fisher Scientific (Loughborough, UK).
C18 columns, Pierce trypsin protease, lysis buffer, Pierce protease inhibitor tablets and lysyl-endopeptidase LysC were purchased from Thermo Scientific (Rockford, IL, USA). Bovine serum albumin and Bradford’s reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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10

Quantitative Protein Profiling by TMT

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The protein concentrations of samples were determined by the bicinchoninic acid protein assay and normalized to the same concentration (1 ug μL−1). For reduction and alkylation, 5 μL de 200 mM Tris (2-carboxyethyl) phosphine was added and incubated at 55 °C for 1 h. Proteins were alkylated with 60 mM iodoacetamide at room temperature for 30 min, protected from light. Samples were subjected to acetone precipitation to purify proteins by incubating with 6 volumes of ice-cold acetone at −20 °C for 4 h. Precipitated proteins were centrifuged at 8000× g for 10 min at 4 °C and the pellet redissolved in 100 μL of 50 mM TEAB (pH 8.5). Proteins were digested by trypsin (Pierce trypsin protease, Thermo Scientific, MA) at a ratio of 1:50 (trypsin:protein, w/w) by incubating overnight at 37 °C.
For quantitative proteomics, each digested protein sample was labelled with an appropriate TMT2plex Isobaric Label Reagent (Thermo Scientific, MA), according to the protocol supplied by the manufacturer. Briefly, the control and treated peptide samples were labelled with reagents TMT-126 and TMT-127, respectively. To quench the TMT reaction, 8 μL of 5% hydroxylamine was added to each sample and incubated for 15 min at room temperature. All the labelled peptides were combined in equal amounts and dried using a speed vacuum system.
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