Modified trypsin

Manufactured by Roche

Sourced in United States

Modified trypsin is a proteolytic enzyme that is commonly used in laboratory settings for the digestion and processing of proteins. It is derived from bovine pancreas and has been chemically or enzymatically modified to enhance its stability and specificity. The primary function of modified trypsin is to cleave peptide bonds within protein substrates, facilitating their analysis and characterization.

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Lab products found in correlation

Mascot, by Matrix Science (2 mentions) Omix c18 tips, by Agilent Technologies (1 mentions) Alliance 2695 hplc, by Waters Corporation (1 mentions) Chymotrypsin, by Roche (1 mentions) Speedvac concentrator, by Thermo Fisher Scientific (1 mentions) C18 ziptip, by Merck Group (1 mentions) Trypsin, by Matrix Science (1 mentions)

Spelling variants of this product

Trypsin (197 citations) Sequencing-grade modified trypsin (10 citations) Modified bovine trypsin (2 citations) Sequence grade-modified trypsin (2 citations)

8 protocols using modified trypsin

Trypsin Digestion and SCX Fractionation

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An
aliquot (400 μg) of MPF suspended in 50 μL of 0.2
M ammonium bicarbonate was digested with 10 μg modified trypsin
(Roche Applied Science) at 37 °C for 4 h, followed by overnight
digestion with an additional 10 μg of trypsin. Samples were
dried under vacuum, reconstituted in H₂O, and dried three
times before suspension in 120 μL of 3% acetonitrile (ACN) containing
0.1% acetic acid. Insoluble material was removed from the sample by
centrifugation at 16 000g for 10 min.
Peptides were separated by strong cation exchange (SCX) chromatography
using an Alliance 2695 HPLC (Waters, Milford, MA) coupled to a PolyLC
polysulfethyl A column (4.6 mm × 100 mm) (The Nest Group, Southboro,
MA) with an increasing linear gradient of KCl (0 to 500 mM) in 10
mM KH₂PO₄, 25% ACN and a flow rate of 1 mL/min.
A total of 12 fractions were collected and dried under vacuum. The
fractions were reconstituted in 100 μL of 0.1% trifluoroacetic
acid and desalted using OMIX C18 tips according to the manufacturer’s
instructions (Varian, Palo Alto, CA). Desalted samples were dried
under vacuum and suspended in 11 μL of 3% ACN, 0.1% formic acid.
Samples were sonicated for 5 min, followed by centrifugation for 10
min at 16 000g and transferred to an autosampler
vial for LC–MS/MS analyses as described below.

Chandler J.C., Sutherland M.D., Harton M.R., Molins C.R., Anderson R.V., Heaslip D.G., Bosio C.M, & Belisle J.T. (2014). Francisella tularensis LVS Surface and Membrane Proteins as Targets of Effective Post-Exposure Immunization for Tularemia. Journal of Proteome Research, 14(2), 664-675.

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TDP-43 Isoform Identification by Mass Spectrometry

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The Sarkosyl-insoluble, SDS/urea-soluble fractions were separated on 4 ~ 20% polyacrylamide gradient gels by SDS-PAGE and immunochemically detected as described26 (link). Briefly, for immunoblotting, mAb anti-TDP-43 (60019-2-Ig, 1:3000, ProteinTech Group), polyclonal anti-TDP (10782-1-AP, 1:3000, ProteinTech Group) and pS409/410 (rabbit polyclonal, 1:1000) were used. Bands of TDP-43 and its derivatives were excised and soaked in 50 mM Tris-HCl, pH 8.0, containing 50% acetonitrile for 30 min. The gel was dried in a Speed-Vac (Savant) and incubated in 50 mM Tris-HCl, pH 8.0 containing 125–250 ng of modified trypsin (Roche Diagnostics, Mannheim, Germany) or chymotrypsin (Roche Diagnostics, Mannheim, Germany) at 37 °C for 6–20 hours. The digests were extracted from the gel twice with 100 μl of 0.1% TFA containing 60% acetonitrile. These two extracts were combined, evaporated in a Speed-Vac, and stored at −80 °C until assayed.

Kametani F., Obi T., Shishido T., Akatsu H., Murayama S., Saito Y., Yoshida M, & Hasegawa M. (2016). Mass spectrometric analysis of accumulated TDP-43 in amyotrophic lateral sclerosis brains. Scientific Reports, 6, 23281.

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Protein Digestion and Peptide Extraction

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Detected protein bands were excised from the gel, transferred to 100 µL of 0.01 M of dithiothreitol/0.1 M of Tris (pH 8.5), and incubated at 55°C for 1 to 2 hours. After cooling, the gel bands were incubated with 100 µL of 0.03 M of iodoacetamide/0.1 M of Tris (pH 8.5) for 30 minutes in the dark, were washed twice with 200 µL of 0.05 M of Tris (pH 8.5)/30% acetonitrile for 20 minutes with shaking, and were then washed once with 100 µL of acetonitrile for 2 to 3 minutes until the gel turned an opaque white. After removing the acetonitrile, the gel pieces were dried for 20 to 30 minutes in a Speed-Vac Concentrator (Thermo Scientific). Gels were digested by adding 0.08 to 0.10 µg of modified trypsin (sequencing grade, Roche Molecular Biochemicals, Indianapolis, IN, USA) in 15 to 20 µL of 0.025 M of Tris (pH 8.5) or enough volume to completely hydrate the gel. The tubes were placed in a heating block at 32°C and left overnight. Peptides were extracted with 50 µL of 50% acetonitrile/2% trifluoroacetic acid, and the combined extracts were either dried or reduced in volume in a Speed-Vac Concentrator to about 10 µL pending further analysis.

Zhu W, & Lee S.W. (2016). Surface interactions between two of the main periodontal pathogens: Porphyromonas gingivalis and Tannerella forsythia. Journal of Periodontal & Implant Science, 46(1), 2-9.

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Protein Identification by MALDI-TOF Mass Spectrometry

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The protein spots that showed more than two time expression difference were excised from the gel and destained for 10 min using 30 mM potassium ferricyanide and 100 mM sodium thiosulfate. Later, repeatedly washed with water and acetonitrile and digested in-gel with modified trypsin (Roche Diagnostics) in 50 mM ammonium bicarbonate. After digestion, peptides were concentrated using C18-ZipTips (Millipore) and eluted directly on the MALDI target in 1 μl of a saturated solution of α-cyanohydroxycinnapinic acid in 50% acetonitrile (vol/vol). Peptides were analyzed using a Voyager STR-DE MALDI-TOF mass spectrometer (Applied Biosystems) operated in reflectron mode at 20 kV accelerating voltage. The MALDI-TOF mass spectra were calibrated internally with known trypsin peaks, and proteins were identified by searching masses of measured peptides against Ralstonia solanacearum proteins in non-redundant protein databases using the peptide mass fingerprint tool in Mascot (Matrix Science) allowing a mass tolerance of 40 ppm.

Raza W., Ling N., Yang L., Huang Q, & Shen Q. (2016). Response of tomato wilt pathogen Ralstonia solanacearum to the volatile organic compounds produced by a biocontrol strain Bacillus amyloliquefaciens SQR-9. Scientific Reports, 6, 24856.

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Nitrated Protein Identification Protocol

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Protein bands of nitrated proteins that differentiate between control and CAN treated were cut out (Figure S2). Destaining, trypsin digestion, and peptide extraction were done as was described by Eckermann et al. (2002) (link). Gel pieces were washed in a mixture of 40% (v/v) acetonitrile and 60% (v/v) 50-mM ammonium bicarbonate for 1–2 h. Destained gel bands were dried by vacuum centrifugation, and modified trypsin (a sequencing grade, Roche) (30 ng µl^-1), dissolved in 50-mM ammonium bicarbonate was added. Trypsin digestion was performed at 37°C (overnight). The gel was incubated in sequence: 1) water, 2) acetonitrile, 3) 5% (v/v) formic acid, and again 4) acetonitrile. After each step, supernatants were collected. The combined supernatant was lyophilized and resolved in a mixture of 10% (v/v) acetonitrile and 90% of 0.1% trifluoroacetic acid.

Staszek P., Krasuska U., Otulak-Kozieł K., Fettke J, & Gniazdowska A. (2019). Canavanine-Induced Decrease in Nitric Oxide Synthesis Alters Activity of Antioxidant System but Does Not Impact S-Nitrosoglutathione Catabolism in Tomato Roots. Frontiers in Plant Science, 10, 1077.

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Mass Spectrometry Analysis of S. pombe Proteins

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Purified proteins (2 µg) were incubated with 10 mM ATPγS in buffer A for 1.5 h at 30°C. After boiling in SDS sample buffer, samples were separated by SDS-PAGE (6% gel). After in-gel digestion with modified trypsin (Roche), the resulting peptides were analysed by online liquid chromatography–tandem mass spectrometry on a Finnigan LCQ Advantage (Thermo Fisher), as previously described [18 (link),32 (link)]. All tandem mass spectra were searched against the S. pombe non-redundant protein database, including common contaminants such as trypsin and keratin, using Mascot (Matrix Science, London, UK).

Akai Y., Kanai R., Nakazawa N., Ebe M., Toyoshima C, & Yanagida M. (2014). ATPase-dependent auto-phosphorylation of the open condensin hinge diminishes DNA binding. Open Biology, 4(12), 140193.

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Tryptic Digestion and Peptide Extraction

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Protein spots of interest were excised from gels and digested with modified trypsin (Roche Molecular Biochemicals, Indianapolis, IN, USA). The gel plugs were washed several times with 100 mM ammonium bicarbonate (NH₄HCO₃) in 50% acetonitrile, the gel pieces were subjected to a reduction step using 10 mM DTT in 100 mM NH₄HCO₃ buffer (45 min at 56°C). Alkylation was performed with a solution of 55 mM iodoacetamide in 100 mM NH₄HCO₃ (30 min at room temperature in the dark) followed by in-gel digestion with 20 μl of trypsin (10 ng/μl) in 50 mM NH₄HCO₃ (overnight at 37°C). Subsequently, the peptides were extracted in NH₄HCO₃ buffer with 5% formic acid. Samples were vacuum-dried and reconstituted in 25 μl of sample preparation solution (98% water, 2% acetonitrile, and 0.5% formic acid).

Devasundaram S., Gopalan A., Das S.D, & Raja A. (2016). Proteomics Analysis of Three Different Strains of Mycobacterium tuberculosis under In vitro Hypoxia and Evaluation of Hypoxia Associated Antigen’s Specific Memory T Cells in Healthy Household Contacts. Frontiers in Microbiology, 7, 1275.

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Trypsin-based Protein Digestion Protocol

Cited 1 time

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Spots from 2-DE were excised and digested with modified trypsin (Roche, Mannheim, Germany) as previously described by Millares et al [13] (link). In brief, gel particles were washed and dehydrated with acetonitrile. Proteins were reduced with DTT, and S-alkylated with iodoacetamide. Gel particles were washed with NH₄HCO₃ and then dried under vacuum rehydrated with the digestion solution. After incubation for 30 min at 4°C, supernatants were replaced by NH₄HCO₃, and gel particles were incubated overnight at 37°C. Trifluoroacetic acid, 2%, was added to end the digestion.

Miao J., Niu J., Wang K., Xiao Y., Du Y., Zhou L., Duan L., Li S., Yang G., Chen L., Tong M, & Miao Y. (2014). Heat Shock Factor 2 Levels Are Associated with the Severity of Ulcerative Colitis. PLoS ONE, 9(2), e88822.

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