Protein Digestion

The APL offers digestion in-gel or in-solutionprotein digestion using protocols that include reduction and alkylation of Cys residues. The protocols describe digestion for trypsin (the most commonly used protease in protein identification), but can be easily adapted for other enzymatic or chemical reagents if different cleavage specificity is needed. These protocols are available to APL clients by request.

Which enzymatic or chemical reagent to choose?

Choosing the right reagent for protein digestion is critical sometimes. Using tools for predicting cleavage sites of proteases or chemicals in a protein sequence may be very helpful in designing your next experiment. For example, Peptide Cutter at Expasy and MS-digest at ProteinProspector simulate digestion using known specificity of proteases and chemicals. Several of them are listed p:

  • Trypsin - preferentially cleaves proteins at the carboxyl group of basic residues, Arg and Lys, except where these residues are directly followed by a Pro residue.
  • Chymotrypsin - preferentially cleaves at the carboxyl group of aromatic residues, Phe, Tyr and Trp. It almost never cleaves at Asp, Glu, Gly or Pro.
  • Pepsin - relatively nonspecific, preferentially cleaves at the carboxyl group of aromatic residues (Phe, Tyr, Trp) and Leu. It does not cleave at Val, Ala or Gly.
  • Endoproteinase Lys-C - cleaves at the carboxyl group of Lys residues and is very effective in gel digests.
  • Endoproteinase Glu-C - prefers cleavage at Glu residues over Asp residues at 100-fold greater rate in all buffers. It cleaves Glu-Pro and Asp-Pro bonds very slowly.
  • Cyanogen bromide - cleaves at the carboxyl group of Met residues. Since this amino acid is relatively infrequent in proteins, this cleavage tends to produce relatively large and few peptides. Met-X amino acid bonds are cleaved specifically and almost quantitatively, with the exception of Met-Ser and Met-Thr bonds, where the hydroxyl group of the neighboring side chain may interfere with ring opening of the iminolactone intermediate.

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