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).
- Which residues does Endoproteinase AspN cut?
- 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.
- I am using Trypsin and am wondering about specificity. Does it cut at additional sites when in high concentration?
- Is there a simple way to remove Trypsin after protein cleavage?
- How does one do a Trypsin in-gel digest?
- 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?
- What is the Proteinase K activity in commonly used buffers?
- O-Glycosidase Application Note 1 (P0733)
- O-Glycosidase (P0733)
- Endo-α-N-Acetylgalactosaminidase Application Note 1
- Removal of terminal N-acetylglucosamine from the biantennary N-linked sugars of IgG
- Typical Reaction Conditions (P0732)
- Typical Reaction Conditions for β1-4 Galactosidase (P0730)
- RNase B Deglycosylation Protocol (P7817)
- Protocol using Trypsin-ultra™, Mass Spectrometry Grade (P8101)
- Trypsin Digestion Protocol using NEB Trypsin-ultra™ and the FASP Kit
- Typical Reaction Conditions for β1-4 Galactosidase S (P0745)
- Typical Reaction Conditions for α2-3 Neuraminidase S (P0743)
- Typical Reaction Conditions for β-N-Acetylglucosaminidase S (P0744)
- Reaction Protocols for Protein Deglycosylation Mix II (P6044)
- In-gel Digestion Protocol for Endoproteinase LysC (P8109)
- Typical Reaction Conditions for Endoproteinase LysC
- Typical GluC Digest Reaction Conditions (P8100)
- Typical GluC In-Gel Digest Reaction Conditions (P8100)
- Typical Trypsin and GluC Co-Digest Reaction Conditions (P8100)
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