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).
Protocols for Proteome Analysis
- Typical Reaction Conditions for β-N-Acetylglucosaminidase S (P0744)
- PNGase F Protocol
- Rapid PNGase F by SDS-PAGE Protocol (P0710)
- Intact Protein LS-ESI-TOF Protocol (P0710)
- Glycoproteomics: Buffer Exchange Protocols (P0710)
- Glycan SPE C18 and Graphitized Carbon Protocols (P0710)
- Rapid PNGase F Protocols (P0710)
- Typical Reaction Conditions for α1-3, 4 Fucosidase (P0769)
- Typical Reaction Conditions for α1-3, 4, 6 Galactosidase (P0747)
- Typical Reaction Conditions for α1-2, 3, 4, 6 Fucosidase (P0748)
- Reaction conditions for Simultaneous Digestion of IgG with IdeZ Protease (IgG-specific) and PNGase F (fragmentation and deglycosylation) (P0770)
- Reaction Conditions for IdeZ Protease (IgG-specific) (P0770)
- Rapid PNGase F (non-reducing format) (P0711) SDS-PAGE Protocol
- Rapid PNGase F (non-reducing format) (P0711) Reaction Protocol
- Glycoproteomics: Buffer Exchange Protocols (P0711)
- Typical Reaction Conditions for Endo F3 Protocol (P0771)
- Typical Reaction Conditions for Endoproteinase LysC
- In-gel Digestion Protocol for Endoproteinase LysC (P8109)
- Typical Reaction Conditions for a1-2,3,6 Mannosidase (P0768)
- Reaction Conditions for PNGase A (P0707)
- Removal of Endo F2 by Magnetic Beads (P0772)
- Endo F2 Reaction Protocol (P0772)
- Endo H/Endo Hf Protocol
- O-Glycosidase Application Note 1 (P0733)
- O-Glycosidase (P0733)
- Western Analysis (E8023)
- RNase B Deglycosylation Protocol (P7817)
- Endo D Removal Magnetic Chitin Bead Protocol (P0742)
- Reaction Conditions for Endo D (P0742)
- Protocol using Trypsin-ultra™, Mass Spectrometry Grade (P8101)
- Endo S Removal Magnetic Chitin Bead Protocol (P0741)
- Remove-iT® PNGase F Magnetic Chitin Bead Protocol (P0706)
- Reaction Conditions for Endo S (P0741)
- Reaction Conditions for Remove-iT® PNGase F (P0706)
- Typical Reaction Conditions α-N-Acetylgalactosaminidase (P0734)
- Typical Trypsin and GluC Co-Digest Reaction Conditions (P8100)
- Typical GluC In-Gel Digest Reaction Conditions (P8100)
- Typical GluC Digest Reaction Conditions (P8100)
- a1-2,4,6 Fucosidase O Digestion of Released Labeled Glycans Protocol
- Typical Reaction Conditions for α2-3,6,8,9 Neuraminidase A (P0722)
- Typical Reaction Conditions for α2-3,6,8 Neuraminidase (P0720)
- Trypsin Digestion Protocol using NEB Trypsin-ultra™ and the FASP Kit
- Reaction Protocols for Protein Deglycosylation Mix II (P6044)
- Endo-α-N-Acetylgalactosaminidase Application Note 1
- Use SNAP-Capture Magnetic Beads (S9145)
The Glycobiology brochure provides information on the suite of endo- and exoglycosidases, and deglycosylation enzymes offered by NEB.
The Glycoproteomics brochure provides information on the suite of endo- and exoglycosidases, and deglycosylation enzymes offered by NEB.
Other Tools & Resources
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