Proteins found in nature vary greatly in size from 5 kDa to greater than 400 kDa. While it is possible to study intact proteins by mass spectrometry (MS) and the modifications present on these proteins, the most common proteomic approaches currently utilize digestion with site-specific proteases to generate smaller fragments, peptides, as a first digestion with site-specific proteases to generate smaller fragments, peptides, as a first step in the analyses (protein digestion). Peptides are easier to characterize and can be separated using reverse phase supports by high performance liquid chromatography (HPLC) using a C18 column. HPLC-coupled to a Tandem MS is used to obtain fragmentation data of individual peptides. This digestion of proteins into smaller pieces is typically carried out by proteases such as trypsin (NEB #P8101) and Endoproteinases GluC (NEB #P8100) and AspN (NEB #P8104).
FAQs for Protein Digestion
- I am using Trypsin and am wondering about specificity. Does it cut at additional sites when in high concentration?
- Which residues does Endoproteinase AspN cut?
- What is the Proteinase K activity in commonly used buffers?
- 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?
- How does one do a Trypsin in-gel digest?
- Is there a simple way to remove Trypsin after protein cleavage?
- Are there any amino acid residues that inhibit or reduce the efficiency of digestion of glutamate residues in a peptide sequence with Endoproteinase GluC? The site I want to digest has a glutamate residue followed by a proline residue and some enzymes are inhibited by the presence of a proline after the hydrolysis site.
Protocols for Protein Digestion
- O-Glycosidase (P0733)
- O-Glycosidase Application Note 1 (P0733)
- Endo-α-N-Acetylgalactosaminidase Application Note 1
- In-gel Digestion Protocol for Endoproteinase LysC (P8109)
- Protocol using Trypsin-ultra™, Mass Spectrometry Grade (P8101)
- Reaction Protocols for Protein Deglycosylation Mix II (P6044)
- Removal of terminal N-acetylglucosamine from the biantennary N-linked sugars of IgG
- RNase B Deglycosylation Protocol (P7817)
- Trypsin Digestion Protocol using NEB Trypsin-ultra™ and the FASP Kit
- Typical GluC Digest Reaction Conditions (P8100)
- Typical GluC In-Gel Digest Reaction Conditions (P8100)
- Typical Reaction Conditions (P0732)
- Typical Reaction Conditions for α2-3 Neuraminidase S (P0743)
- Typical Reaction Conditions for β-N-Acetylglucosaminidase S (P0744)
- Typical Reaction Conditions for β1-4 Galactosidase S (P0745)
- Typical Reaction Conditions for β1-4 Galactosidase (P0730)
- Typical Reaction Conditions for Endoproteinase LysC (P8109)
- Typical Trypsin and GluC Co-Digest Reaction Conditions (P8100)
The Glycobiology brochure provides information on the suite of endo- and exoglycosidases, and deglycosylation enzymes offered by NEB.
Other Tools & Resources
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