Direct comparison
Trypsin vs Chymotrypsin: Cleavage Sites Compared
Trypsin vs chymotrypsin: P1 cleavage residues, the proline block, specificity tiers and TPCK-treated trypsin, with sources.
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How do Trypsin, Chymotrypsin compare side by side?
The table below compares Trypsin, Chymotrypsin across 11 procurement-relevant dimensions, from cleavage position through source.
Side-by-side comparison
| Dimension | Trypsin | Chymotrypsin |
|---|---|---|
| Cleavage position | Carboxyl (C-terminal) side of the recognised residue (Promega V5111). | C-terminal side of the recognised residue (Expasy: "C-term to" the listed residues). |
| Residue at P1 | Lysine (K) or arginine (R). | High specificity: phenylalanine (F), tyrosine (Y) or tryptophan (W). Low specificity adds leucine (L), methionine (M) and histidine (H). |
| Proline rule (P1') | Proline at P1' usually blocks cleavage. | Proline at P1' nearly fully blocks cleavage whatever residue is at P1. |
| Specificity tiers | One rule (K or R, not before P), with a short list of exceptions. | Two rule sets in Expasy PeptideCutter: high specificity (F, Y, W) and low specificity (F, L, Y, W, M, H). |
| Listed exceptions (no cleavage) | Expasy lists context exceptions including: C or D at P2 with K and D; C with K and H or Y; C with R and K; R with R and H or R. | High specificity: W followed by M or P is not cut. Low specificity: M not before P or Y; H not before D, M, P or W. |
| Relationship in enzyme preparations | Trypsin preparations can carry chymotryptic activity; TPCK treatment "irreversibly inactivates chymotrypsin by blocking its active site" (Promega V5111). | TPCK irreversibly inactivates it, which is why it is used to strip chymotryptic activity from trypsin preparations. |
| Protecting the enzyme from self-digestion | Reductive methylation of lysines blocks autolysis while keeping activity (Promega V5111, Sequencing Grade Modified Trypsin). | — |
| Typical working conditions (trypsin) | Enzyme to substrate typically 1:100 to 1:20 w/w; maximum activity at pH 7 to 9, reversibly inactivated at pH 4 (Promega V5111). Research use only. | — |
| Role in a proteomics digest | The protease whose K/R rule defines the peptides in a standard digest. | Used as an additional protease to reach sequence space trypsin cannot, at the cost of extra runs (CASRAI mass spectrometry proteomics guide). |
| When to pick each | Pick trypsin when the cleavage sites you need follow K or R. | Pick chymotrypsin when you need cleavage after F, Y or W (or, less specifically, L, M or H), as a second protease alongside trypsin. |
| Source | Expasy PeptideCutter enzyme rules; Promega Sequencing Grade Modified Trypsin V5111 (both read 2026-10-29). | Expasy PeptideCutter enzyme rules (read 2026-10-29); Promega V5111 for the TPCK statement. |
Common questions
Common questions about Trypsin vs Chymotrypsin
What is the difference between trypsin and chymotrypsin?
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They are both proteases, but they cut after different residues. Trypsin cleaves on the carboxyl side of lysine and arginine; chymotrypsin cleaves after phenylalanine, tyrosine and tryptophan (high specificity) and, less specifically, after leucine, methionine and histidine. A proline right after the cut site blocks both (Expasy PeptideCutter, read 2026-10-29).
