Proteomics using digestion of proteins followed by analysis by mass spectrometry has found wide acceptance as a way to investigate cellular expression and control. The most common protease used for digestion of proteins to produce peptides in simple or complex mixtures is Trypsin (NEB #P8101). Trypsin is treated with TPCK to destroy chymotryptic activity and acetylated on Lysine residues to prevent autolysis. Trypsin digestion produces peptides that have two positive charges in acidic solutions: the N-terminal amino group and at the C-terminal of Lysine or Arginine on the side chain. These doubly charged peptides are fragmented by Collision-Induced Disassociation (CID) and produce fingerprint spectra in a tandem mass spectrometer. A program such as Sequest or Mascot is then used to compare these spectra to the in silico predicted spectra to find the best matches if any.
Other proteases such as the Endoproteinases GluC (NEB #P8100), AspN (NEB #P8104), LysC and ArgC are also used to generate peptides that can be used in the same way. These other specificities can be used to generate larger peptides with more charges per peptide or to isolate a specific peptide when looking for a specific modification.
Trypsin-digested BSA MS Standard (CAM-modified) (NEB #P8108) is a complex mixture of peptides produced by Trypsin digestion of Bovine Serum Albumin (BSA) that was reduced and alkylated with Iodoacetamide (CAM-modified). This peptide mixture can be used to test a Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF), test a chromatographic separation or Electrospray Ionization (ESI) mass spectrometer (TOF, Q-TOF or Ion Trap).
- O-Glycosidase Application Note 1 (P0733)
- O-Glycosidase (P0733)
- Western Analysis (E8023)
- Endo-α-N-Acetylgalactosaminidase Application Note 1
- Typical Reaction Conditions for α2-3,6,8 Neuraminidase (P0720)
- Use SNAP-Capture Magnetic Beads (S9145)
- Endo H/Endo Hf Protocol
- Typical Reaction Conditions α-N-Acetylgalactosaminidase (P0734)
- Reaction Conditions for Remove-iT® PNGase F (P0706)
- Reaction Conditions for Endo S (P0741)
- Endo S Removal Magnetic Chitin Bead Protocol (P0741)
- Remove-iT® PNGase F Magnetic Chitin Bead Protocol (P0706)
- Reaction Conditions for Endo D (P0742)
- Endo D Removal Magnetic Chitin Bead Protocol (P0742)
- RNase B Deglycosylation Protocol (P7817)
- Protocol using Trypsin-ultra™, Mass Spectrometry Grade (P8101)
- Trypsin Digestion Protocol using NEB Trypsin-ultra™ and the FASP Kit
- PNGase F Protocol
- Typical Reaction Conditions for α2-3,6,8,9 Neuraminidase A (P0722)
- Typical Reaction Conditions for β-N-Acetylglucosaminidase S (P0744)
- Rapid PNGase F Protocols (P0710)
- Intact Protein LS-ESI-TOF Protocol (P0710)
- Rapid PNGase F by SDS-PAGE Protocol (P0710)
- Glycan SPE C18 and Graphitized Carbon Protocols (P0710)
- Glycoproteomics: Buffer Exchange Protocols (P0710)
- Typical Reaction Conditions for α1-3, 4, 6 Galactosidase (P0747)
- Typical Reaction Conditions for α1-2, 3, 4, 6 Fucosidase (P0748)
- Typical Reaction Conditions for α1-3, 4 Fucosidase (P0769)
- Reaction Conditions for IdeZ Protease (IgG-specific) (P0770)
- Reaction conditions for Simultaneous Digestion of IgG with IdeZ Protease (IgG-specific) and PNGase F (fragmentation and deglycosylation) (P0770)
- Glycoproteomics: Buffer Exchange Protocols (P0711)
- Rapid PNGase F (non-reducing format) (P0711) Reaction Protocol
- Rapid PNGase F (non-reducing format) (P0711) SDS-PAGE Protocol
- Reaction Protocols for Protein Deglycosylation Mix II (P6044)
- Typical Reaction Conditions for Endo F3 Protocol (P0771)
- Reaction Conditions for PNGase A (P0707)
- Typical Reaction Conditions for a1-2,3,6 Mannosidase (P0768)
- In-gel Digestion Protocol for Endoproteinase LysC (P8109)
- Typical Reaction Conditions for Endoproteinase LysC
- Endo F2 Reaction Protocol (P0772)
- Removal of Endo F2 by Magnetic Beads (P0772)
- Typical GluC Digest Reaction Conditions (P8100)
- Typical GluC In-Gel Digest Reaction Conditions (P8100)
- Typical Trypsin and GluC Co-Digest Reaction Conditions (P8100)
- a1-2,4,6 Fucosidase O Digestion of Released Labeled Glycans Protocol
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