Sequencing grade trypsin

Manufactured by Worthington

Sequencing-grade trypsin is a proteolytic enzyme used in the preparation of protein samples for mass spectrometry and other analytical techniques. It is specifically formulated to minimize the introduction of contaminants during protein digestion, ensuring accurate and reliable results in protein sequencing and identification.

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Lab products found in correlation

Reprosil gold 120 c18, by Dr. Maisch (2 mentions) Easy nlc 1000, by Thermo Fisher Scientific (2 mentions) Picotip emitter, by New Objective (2 mentions) Dithiothreitol (dtt), by Merck Group (2 mentions) Iodoacetamide, by Merck Group (2 mentions) Q exactive hf orbitrap, by Thermo Fisher Scientific (1 mentions) Orbitrap exploris 480, by Thermo Fisher Scientific (1 mentions) Sodium hydroxide (naoh), by Merck Group (1 mentions) Trypsin, by Worthington (1 mentions)

Spelling variants of this product

Trypsin (192 citations) Sequencing grade TPCK-trypsin (3 citations) Sequencing grade modified trypsin (2 citations)

3 protocols using sequencing grade trypsin

In Vitro Peptide Methylation Assay

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The in vitro assay was carried out using either full-length peptide 3, the C-terminal 38-mer of peptide 3, or full-length peptide 5. A 50-μL reaction was set up anaerobically in 50 mM Hepes and 100 mM NaCl, pH 7.6, containing 20 to 180 μM substrate, 4 to 20 μM enzyme (the case by case concentrations are specified in SI Appendix), and 1 to 2 mM SAM or d₃-SAM. The reaction was initiated by adding 2 mM Ti(III) citrate (final concentration). The reaction proceeded in the dark for 16 h. A 10-μL aliquot was taken out from the anaerobic chamber for analysis with MALDI-TOF MS after cleanup with a Ziptip C18 (Agilent Technologies). If the reaction conversion was satisfactory, the remainder of the reaction was taken out from the anaerobic chamber, diluted with an equal volume of 50 mM ammonium bicarbonate, and digested with 2 μg of sequencing grade trypsin (Worthington) at 37 °C for 8 h. After the digestion, the reaction was acidified with 1% formic acid and cleaned up with a TopTip C18 column (Glygen). Tryptic fragments were eluted with 60% acetonitrile + 0.1% formic acid. The elution was lyophilized and redissolved in H₂O for subsequent LC-ESI-HRMS analysis.

Yu Y, & van der Donk W.A. (2022). Biosynthesis of 3-thia-α-amino acids on a carrier peptide. Proceedings of the National Academy of Sciences of the United States of America, 119(29), e2205285119.

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Mass Spectrometry Proteomics Workflow

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Resin containing purified immunoprecipitates was incubated in 200 mM HEPES (4-(hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.5 containing 5 mM dithiothreitol (Sigma-Aldrich) at 37°C for 1 hr, followed by alkylation of cysteine residues using 15 mM iodoacetamide (Sigma-Aldrich) in the dark at room temperature for 1 hour. Alkylated proteins were diluted in 1:6 ratio (v/v) in ultrapure water prior to digestion using sequencing-grade trypsin (Worthington Biochemical Corp) at 37°C for 16 hrs. Digested peptides were subsequently desalted using self-packed C18 Stage Tips (3M Empore™) (Rappsilber et al., 2003) for LC-MS/MS analysis. Desalted peptides were resuspended in 0.1% (v/v) formic acid and analyzed on an Easy-nLC 1000 (Thermo Fisher Scientific) coupled to Orbitrap Q-Exactive HF (Thermo Fisher Scientific) mass spectrometer. Chromatography for peptide separation was performed using increasing organic proportion of acetonitrile (5–40 % (v/v)) on a self-packed analytical column using PicoTip™ emitter (New Objective, Woburn, MA) containing Reprosil Gold 120 C18, 1.9 um particle size resin (Dr. Maisch, Ammerbuch-Entringen, Germany) over a 120-min gradient at a flow rate of 300 nl/min. The mass spectrometry analyzer was operated in data dependent acquisition mode with a top ten method at a mass range of 300 – 2000 Da.

Chung J., Wu X., Lambert T.J., Lai Z.W., Walther T.C, & Farese RV J.r. (2019). Lipid droplet assembly factor-1 and seipin form a lipid droplet assembly complex. Developmental cell, 51(5), 551-563.e7.

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Proteomic Analysis of Lipid-Enriched Fractions

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Proteins pellets from LD-enriched fractions were resuspended in 0.1 M NaOH (Sigma-Aldrich) and subsequently neutralized using 200 mM HEPES. Solubilized proteins were reduced using 5 mM dithiothreitol (Sigma-Aldrich), pH 7.5, at 37 °C for 1 h. Reduced disulfide bonds of cysteine residues were alkylated using 15 mM iodoacetamide (Sigma-Aldrich) for 1 h in the dark. Excessive iodoacetamide was quenched using 10 mM dithiothreitol. The alkylated protein mixture was diluted six-fold (v/v) using 20 mM HEPES, pH 7.5, and digested for 16 h at 37 °C with sequencing-grade trypsin (Worthington Biochemical) in a 1:100 trypsin-to-protein ratio. Digested peptides were de-salted using self-packed C18 STAGE tips (3 M Empore)^{78 (link)}. De-salted peptides were dissolved in 0.1% (v/v) formic acid and injected onto an Easy-nLC 1000 (Thermo Fisher Scientific), coupled to an Orbitrap Exploris 480 (Thermo Fisher Scientific). Peptide separation was performed on a 500-mm self-packed analytical column using PicoTip emitter (New Objective) containing Reprosil Gold 120 C-18, 1.9-µm particle-size resin (Dr. Maisch). Chromatography separation was carried out using increasing organic proportion of acetonitrile (5–40 % (v/v)) containing 0.1 % (v/v) formic acid over a 120 min gradient at a flow rate of 300 nl min⁻¹.

Song J., Mizrak A., Lee C.W., Cicconet M., Lai Z.W., Tang W.C., Lu C.H., Mohr S.E., Farese RV J.r, & Walther T.C. (2022). Identification of two pathways mediating protein targeting from ER to lipid droplets. Nature Cell Biology, 24(9), 1364-1377.

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