Trypsin-ultra™, Mass Spectrometry Grade

Description

Trypsin-ultra™, Mass Spectrometry Grade is a serine endopeptidase. It selectively cleaves peptide bonds C-terminal to lysine and arginine residues (1). Trypsin-ultra is treated with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK) to inactivate any remaining chymotryptic activity. It is modified by acetylation of the ε-amino groups of lysine residues to prevent autolysis. Trypsin-ultra (TPCKtreated) cleaves at Lys-Pro and Arg-Pro bonds at a much slower rate than other amino acid residues (2).


Digestion with Trypsin-ultra results in a high number of proteins identified, and sequence coverages as high as 90%

A 3-hour Trypsin-ultra digest of Pyrococcus furiosus using a 1:50 enzyme:substrate ratio, followed by ESI-MS, resulted in 418 proteins identified with individual protein sequence coverage as high as 90%.

Product Source

Isolated from bovine (Bos taurus) pancreas

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
Trypsin-ultra, Reaction Buffer-202X

Advantages and Features

Applications

  • Digestion of proteins for proteomic analysis by Mass Spectrometry
  • Protein and peptide identification

Properties and Usage

Reaction Conditions

1X Trypsin-ultra, Reaction Buffer
Incubate at 37°C

1X Trypsin-ultra, Reaction Buffer:
50 mM Tris-HCl
20 mM CaCl2
pH 8 @ 25°C

Usage Concentration

100 ng/μl

Storage Temperature

-20°C

Molecular Weight

Theoretical: 23675 daltons

Specific Activity

2.1 μmol/min/mg/

Notes

  1. Substrate must be in phosphate-free buffer to prevent calcium precipitation with both reconstituted enzyme and enzyme buffer.
  2. Trypsin-ultra, Mass Spectrometry Grade is acetylated on multiple lysine residues. This protein appears as a single band on SDS-PAGE. This sequence is also available at www.neb.com.
  3. Reconstitution: Trypsin-ultra, Mass Spectrometry Grade should be reconstituted by the addition of 20–200 μl of high purity water. Rapid autolysis is a function of enzyme concentration.
  4. Storage: Supplied freeze-dried from a sodium acetate and calcium chloride buffer. Store at -20°C.
    Can be stored frozen in solution at -20°C for up to 2 weeks. A decrease in activity will occur if stored in solution. Use only freshly reconstituted protease for best results.

References

  1. Northrop, J.H. and Kunitz, M. (1931). Isolation of protein crystals possessing tryptic activity. Science. 73
  2. Perona, J.J. and Craik, C.S. (1995). Structural basis of substrate specificity in the serine proteases. Protein Sci.. 4, 337-360.

FAQs

  1. Is there a compatible buffer for digesting with Trypsin (P8101S) and Endoproteinase GluC (P8100S) together?
  2. I have a very low concentration of protein and would prefer not to denature as a separate step with buffer exchange before digestion. What denaturants can he use in the Trypsin reaction itself?
  3. I would like to use Trypsin to digest some proteins which are present in a cell culture supernatant. The supernatant is composed of DMEM supplemented with 0.2% BSA and antibiotics. Will the enzyme work in the cellular supernatant? The final reaction volume will be 100-300 ul.
  4. I am using Trypsin and am wondering about specificity. Does it cut at additional sites when in high concentration?
  5. I want to know if calcium can be left out of the buffer since it is causing my DNA-protein complex to precipitate.
  6. I want to use Trypsin on 20 ug of protein. How much enzyme do I need to use?
  7. What is the amino acid sequence Trypsin-ultra?
  8. Will Trypsin work in 50mM Ammonium Bicarbonate, pH 8.3 ?
  9. How does one do a Trypsin in-gel digest?
  10. Is there a simple way to remove Trypsin after protein cleavage?

Protocols

  1. Protocol using Trypsin-ultra™, Mass Spectrometry Grade (P8101)
  2. Trypsin Digestion Protocol using NEB Trypsin-ultra™ and the FASP Kit

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Selection Charts

Interactive Tools

Application Notes

Quality Control

Quality Assurance Statement

  • Modified Trypsin is free of glycerol and detergents which may interfere with Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) Mass Spectrometry (MS) or liquid chromatography (LC) methods.

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Functional Test (Calcitonin Peptide Digestion, LC-MS):
    The endoproteinase is tested for its ability to digest a modified Human calcitonin peptide as determined by LC-MS analysis.
  • Functional Test (Cytochrome C Digestion, LC-MS):
    The endoproteinase is tested for its ability to digest Cytochrome C as determined by LC-MS analysis.
  • Specific Activity (Fluorometric):
    The endoproteinase is tested in a reaction with labeled peptide and the initial rate of digestion is determined by measurement of the increase in fluorescence

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.