Tryptic digestion

Manufactured by Promega

Sourced in United States

Tryptic digestion is a laboratory technique used to cleave, or break down, proteins into smaller peptide fragments. Trypsin, a proteolytic enzyme, is used to catalyze this process. The resulting peptides can then be further analyzed using various analytical methods.

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Lab products found in correlation

Irt peptide, by Biognosys (5 mentions) C18 reverse phase column, by Harvard Apparatus (5 mentions) Iodoacetamide, by Merck Group (5 mentions) Trifluoacetic acid, by Merck Group (3 mentions) Bradford assay, by Bio-Rad (3 mentions) Lys c, by Fujifilm (2 mentions) Rapigest, by Waters Corporation (2 mentions) Non immune mouse igg, by Merck Group (2 mentions) Protein g sepharose, by Cytiva (2 mentions) Tripletof 6600 mass spectrometer, by AB Sciex (1 mentions) Nanolc system, by AB Sciex (1 mentions) Chromxp c18 cl, by AB Sciex (1 mentions) Savant spd 111v speedvac concentrator, by Thermo Fisher Scientific (1 mentions) Tmt 6plex, by Thermo Fisher Scientific (1 mentions) Trifluoroacetic acid, by Merck Group (1 mentions) C18 cartridge, by Waters Corporation (1 mentions) Microson xl sonicator, by Bioventus (1 mentions) Quantitative colorimetric peptide assay kit, by Thermo Fisher Scientific (1 mentions) Rapigest, by Merck Group (1 mentions)

Close spelling variants of this product

Tryptic digestion (sequencing grade (2 citations)

11 protocols using tryptic digestion

Proteomics and Metabolomics of Morphine Exposure

Cited 1 time

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For proteomics, 10 µg of proteins were reduced using TCEP for each sample of cells and EVs at morphine concentrations of 0, 1, 10, 25, 50 and 100 μM (final concentration 5 mM, 30 min, 37 °C) (Sigma-Aldrich, St. Louis, MO, USA), alkylated using iodoacetamide (final concentration 15 mM, 60 min, RT, in dark conditions) (Sigma-Aldrich, St. Louis, MO, USA) and digested by an overnight tryptic digestion (w/w ratio 1:50) (Promega, Madison, WI, USA). The RapiGest surfactant was cleaved by incubating samples with 0.5% trifluoacetic acid (Sigma-Aldrich, St. Louis, MO, USA) for 45 min at 37 °C. Samples were then desalted on a C18 reverse phase column (Harvard Apparatus, Holliston, MA, USA), peptides were dried under vacuum and subsequently resuspended in 5% ACN 0.1% FA (peptides final concentration of 0.5 µg/µL and spiked with iRT peptide (Biognosys, Schlieren, Switzerland) (1:20)).
For metabolomics, cell samples (morphine exposure: 0, 5 and 50 μM) were prepared based on Meister et al. [15 (link)] work, as described in Supplementary File S1.

Vujić T., Schvartz D., Furlani I.L., Meister I., González-Ruiz V., Rudaz S, & Sanchez J.C. (2022). Oxidative Stress and Extracellular Matrix Remodeling Are Signature Pathways of Extracellular Vesicles Released upon Morphine Exposure on Human Brain Microvascular Endothelial Cells. Cells, 11(23), 3926.

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Protein Reduction, Alkylation and Tryptic Digestion

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For each sample, 5 µg proteins was reduced using TCEP (final concentration 5 mM, 30 min, 37 °C) (Sigma-Aldrich, St. Louis, MO, USA), alkylated using iodoacetamide (final concentration 15 mM, 60 min, RT, in dark condition) (Sigma-Aldrich, St. Louis, MO, USA) and digested by an overnight tryptic digestion (w/w ratio 1:50) (Promega, Madison, WI, USA). The RapiGest surfactant was cleaved by incubating samples with 0.5% trifluoacetic acid (Sigma-Aldrich, St. Louis, MO, USA) (45 min, 37 °C). Samples were then desalted on a C18 reverse phase column (Harvard Apparatus, Holliston, MA, USA), peptides were dried under vacuum and subsequently resuspended in 5% ACN 0.1% FA (peptides final concentration of 0.5 µg/µL and spiked with iRT peptide (Biognosys, Schlieren, Switzerland) (1:20)).

Vujić T., Schvartz D., Iliuk A, & Sanchez J.C. (2021). Ubiquinone Metabolism and Transcription HIF-1 Targets Pathway Are Toxicity Signature Pathways Present in Extracellular Vesicles of Paraquat-Exposed Human Brain Microvascular Endothelial Cells. International Journal of Molecular Sciences, 22(10), 5065.

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Protein Reduction, Alkylation, and Digestion

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One milligram of proteins from tissues, cells, or standard glycoproteins, including bovine serum fetuin (Millipore Sigma) and bovine pancreas RNase B (Millipore Sigma), were reduced and alkylated with 5 mM dithiothreitol (37 °C, 1 hour) and 10 mM iodoacetamide (25 °C, 45 minutes, in the dark), respectively. The denatured proteins were first digested by Lys-C (FUJIFILM Wako Chemicals USA. Corporation) in an enzyme to substrate ratio of 1:50 for 2 hours at 25 °C, followed by tryptic digestion (Promega) in an enzyme to substrate ratio of 1:50 for 14 hours at 25 °C. The enzymatic reaction was terminated by adding 50% of formic acid (final pH <3). The reaction solution was then centrifuged at 16,000 × g for 10 minutes at 4 °C.

Cao L., Lih T.M., Hu Y., Schnaubelt M., Chen S.Y., Zhou Y., Guo C., Dong M., Yang W., Eguez R.V., Chen L., Clark D.J., Sodhi A., Li Q.K, & Zhang H. (2022). Characterization of core fucosylation via sequential enzymatic treatments of intact glycopeptides and mass spectrometry analysis. Nature Communications, 13, 3910.

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Fc-part N-Glycosylation Analysis by LC-MS/MS

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N-glycosylation pattern of the Fc-part was analysed by peptide mapping using liquid chromatography/mass spectroscopy (LC/MS/MS), as described previously (6 (link)). Briefly, Etanercept and sTNFR-Fc dissolved in 0.5 M Tris–HCl, 8 M guanidine–HCl and 5 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.6) was reduced with dithiothreitol and carboxy-methylated with sodium monoiodoacetamide. Desalted samples were employed for tryptic digestion (Promega) at 37°C for 4 h. The tryptic digests were dissolved in distilled water containing 2% acetonitrile and 0.1% trifluoroacetic acid. The samples were separated using an Eksigent Nano LC System (SCIEX) using a Nano LC column (3 μm, ChromXP C18CL; SCIEX). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in 90% acetonitrile (solvent B). The chromatography was performed with a gradient from 0% to 55% solvent B for 40 min at a flow rate of 0.3 ml/min. Mass spectrometric analyses were performed by using a TripleTOF 6,600 mass spectrometer (SCIEX). Mass spectra were acquired over m/z 400–2,000 for mass spectrometry (MS) and m/z 100–2,000 for MS/MS. The areas of the peaks were integrated to calculate the relative abundance of each N-glycan.

Kiyoshi M., Tatematsu K.I., Tada M., Sezutsu H., Shibata H, & Ishii-Watabe A. (2020). Structural insight and stability of TNFR-Fc fusion protein (Etanercept) produced by using transgenic silkworms. Journal of Biochemistry, 169(1), 25-33.

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Protein Preparation for Mass Spectrometry

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Each sample (plasma samples (1 µg) and EV samples (70 µL)) was reduced using TCEP 0.1 M (final concentration 5 mM, 30 min, 37 °C) (Sigma-Aldrich), alkylated using iodoacetamide 150 mM (final concentration 15 mM, 60 min, RT, in dark condition) (Sigma-Aldrich), and digested by an overnight tryptic digestion (w/w ratio 1:50, 37 °C) (Promega). The RapiGest surfactant was cleaved by incubating samples with 0.5% trifluoacetic acid (45 min, 37 °C) (Sigma-Aldrich). Samples were desalted on a C18 reverse phase column (Harvard Apparatus) and the remaining peptides were dried in Savant SPD111V SpeedVac Concentrator (Thermo Fisher). They were stored at − 80 °C and, prior to MS injection, they were resuspended in 5% ACN 0.1% FA with the addition of iRT peptides (ratio 1:20) (Biognosys).

Reymond S., Gruaz L, & Sanchez J.C. (2023). Depletion of abundant plasma proteins for extracellular vesicle proteome characterization: benefits and pitfalls. Analytical and Bioanalytical Chemistry, 415(16), 3177-3187.

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Comparative Proteomic Analysis of Mastitis

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100 µg of protein sample was taken from pooled (n = 10) healthy, sub-clinical, and clinical mastitis samples and dissolved in dissolution buffer (0.5 M triethylammonium-bicarbonate, pH 8.5), denatured with 2% SDS followed by reduction of protein using 50 mM tris-(2 carboxyethyl) phosphine (TCEP) at 60 °C for 1 h. Alkylation of cysteine residues was performed using 20 mm IAA in the dark for 30 min followed by tryptic digestion (Promega, 1:20) at 37 °C overnight. The peptide derived from healthy, sub-clinical and clinical samples were labelled with 126, 127, or 128 TMT reagent respectively using TMT 6 Plex (Thermo) according to the manufacturer’s protocol. Peptides were labelled with respective tags and incubated for 2 h, quenched and vacuum centrifuged to dryness.

Bathla S., Sindhu A., Kumar S., Dubey S.K., Pattnaik S., Rawat P., Chopra A., Dang A., Kaushik J.K, & Mohanty A.K. (2020). Tandem Mass Tag (TMT)-based quantitative proteomics reveals potential targets associated with onset of Sub-clinical Mastitis in cows. Scientific Reports, 10, 9321.

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Protein Extraction and Tryptic Digestion

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Cell pellets were resuspended in 0.1% RapiGest (Waters) and 100 mM TEAB (Sigma-Aldrich) and sonicated (five cycles of 20 s with breaks, on ice) and incubated for 10 min at 80 °C. Samples were then spun down (14000 g, 10 min, 4 °C), and the supernatant was recovered. The protein content was measured using Bradford assay (BioRad). For each sample, 10 μg of proteins was reduced using TCEP (final concentration 5 mM, 30 min, 37 °C) (Sigma-Aldrich), alkylated using iodoacetamide (final concentration 15 mM, 60 min, at room temperature, in dark condition) (Sigma-Aldrich), and submitted to an overnight tryptic digestion (w/w ratio 1:50) (Promega). The RapiGest surfactant was cleaved by incubating samples with 1% trifluoroacetic acid (Sigma-Aldrich) (45 min, 37 °C). Samples were then desalted on a C18 reverse phase column (Harvard Apparatus); peptides were dried under vacuum and subsequently resuspended in an appropriate volume of 5% acetonitrile 0.1% formic acid and spiked with iRT peptide (Biognosys, 1:20) for mass spectrometry (MS) analysis.

Pamies D., Vujić T., Schvartz D., Boccard J., Repond C., Nunes C., Rudaz S., Sanchez J.C., González-Ruiz V, & Zurich M.G. (2022). Digoxin Induces Human Astrocyte Reaction In Vitro. Molecular Neurobiology, 60(1), 84-97.

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Proteomic Analysis of FGF1 and R1MAb2 Signaling

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CAL-120 cells were harvested and lysed in 20 mM Hepes at pH 8.0, containing 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, and 1 mM β-glycerophosphate. Whole cell lysates from PBS-treated, FGF1-treated (20 min), and R1MAb2-treated (1 h) CAL-120 cells (3 bioreplicates per condition, total of nine samples) were digested with trypsin and prepared for proteomic analysis. Samples were sonicated using a Misonix Microson XL sonicator followed by centrifugation at 20,000g for 20 min at 15 °C. Protein concentration was determined using a Bradford assay (Bio-Rad). Samples were reduced in 5 mM DTT at 37 °C for 1 h followed by alkylation with 15 mM iodoacetamide at room temperature for 20 min in the dark. Proteins were subjected to a serial digestion using Lys-C (Wako) at an enzyme:substrate (E:S) ratio of 1:50 at 37 °C for 4 h followed by tryptic digestion (Promega) at an E:S ratio of 1:50 at 37 °C overnight in 2 M urea. The peptide mixture was acidified with 20% TFA and desalted using C₁₈ cartridge (500 mg absorbent) from Waters. Peptides were eluted with 3 × 2.0 ml of 60% acetonitrile (ACN)/0.1% TFA followed by peptide concentration measurement using a quantitative colorimetric peptide assay kit (Thermo Fisher Scientific). Equal amounts of peptides per condition (17 mg) were aliquoted and lyophilized overnight.

Chan J., Chan J., Shao L., Stawicki S.S., Pham V.C., Akita R.W., Hafner M., Crocker L., Yu K., Koerber J.T., Schaefer G, & Comps-Agrar L. (2022). Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action. The Journal of Biological Chemistry, 299(1), 102729.

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Platelet Proteome Identification by Mass Spectrometry

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Human platelets (5 × 10⁹ cells/10 ml) were washed with cold PBS and lysed in buffer containing 20 mM HEPES pH 7.40, 150 mM NaCl, 5 mM EDTA, 1% Brij 99, and protease inhibitors (HALTS, Pierce). After 30 min at 4 °C, insoluble material was removed by centrifugation at 16,000 ×g (15 min, 4 °C). The platelet lysates were pre-cleared by adding 20 μg of non-immune mouse IgG (Sigma) and 200 μl of Protein G-Sepharose (Amersham Pharmacia Biotech) and rocking gently at 4 °C for 60 min. Immunoprecipitation was performed by combining 20 μg of the anti-TSP-1 mouse monoclonal MA-IV and 200 μl of Protein G-Sepharose. The samples were incubated for 16 h at 4 °C with gentle rocking. Immune complexes were collected by centrifugation, washed four times in lysis buffer, and separated by SDS-PAGE in the presence of a reducing agent. Coomassie Blue stained bands were subjected to in-gel reduction, carboxyamidomethylation and tryptic digestion (Promega). Multiple peptide sequences were determined in a single run by microcapillary reverse-phase chromatography which was directly coupled to a Finnigan LCQ quadrupole ion trap mass spectrometer equipped with a custom nano-electrospray source. The Harvard Microchemistry Facility completed this analysis on a fee-for-service basis (Miao et al., 2001b (link)).

Duquette M., Nadler M., Okuhara D., Thompson J., Shuttleworth T, & Lawler J. (2014). Members of the thrombospondin gene family bind stromal interaction molecule 1 and regulate calcium channel activity. Matrix biology : journal of the International Society for Matrix Biology, 37, 15-24.

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Protein Extraction and Digestion for Mass Spectrometry

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Cell pellets were resuspended in 0.1% RapiGest (Waters) and 100 mM TEAB (Sigma Aldrich), sonicated (five cycles of 20 s with breaks on ice), and incubated for 10 min at 80 °C. Samples were then spun down (14,000g, 10 min, 4 °C) and the supernatant was recovered. The protein content was measured using Bradford assay (BioRad).
For each sample, 20 μg proteins was reduced using TCEP (final concentration 5 mM, 30 min, 37 °C) (Sigma Aldrich), alkylated using iodoacetamide (final concentration 15 mM, 60 min, RT, in dark condition) (Sigma Aldrich) and digested by an overnight tryptic digestion (w/w ratio 1:50) (Promega). The RapiGest surfactant was cleaved by incubating samples with 0.5% trifluoacetic acid (Sigma Aldrich) (45 min, 37 °C). Samples were then desalted on a C18 reverse phase columns (Harvard Apparatus), peptides were dried under vaccum and subsequently resuspended in 5% ACN 0.1% FA (peptides final concentration of 0.5 μg/μL and spiked with iRT peptide (Biognosys) (1:20)).

Tatjana V., Domitille S, & Jean-Charles S. (2021). Paraquat-induced cholesterol biosynthesis proteins dysregulation in human brain microvascular endothelial cells. Scientific Reports, 11, 18137.

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