2. Enzyme definitions

All default available enzymes (enzymes_definition.py) are listed bellow.

For each of them, there is the equivalent in RPG grammar.

In the following, nomenclature of Schechter and Berger is used. Amino acids before the cleavage site are designated as P1, P2, P3, etc in the N-terminal direction, and as P1’, P2’, P3’, etc in the C-terminal direction. For example, with cleavage site represented as ‘|’:

...P3-P2-P1-|-P1'-P2'-P3'...

In RPG, this nomenclature is represented as:

...(P3)(P2)(P1)(,)(P1')(P2')(P3')...

2.1. Available enzymes

1: Arg-C 2: Asp-N 3: BNPS-Skatole
4: Bromelain 5: Caspase 1 6: Caspase 2
7: Caspase 3 8: Caspase 4 9: Caspase 5
10: Caspase 6 11: Caspase 7 12: Caspase 8
13: Caspase 9 14: Caspase 10 15: Chymotrypsin high specificity
16: Chymotrypsin low specificity 17: Clostripain 18: CNBr
19: Enterokinase 20: Factor Xa 21: Ficin
22: Formic acid 23: Glu-C 24: Glutamyl endopeptidase
25: Granzyme B 26: Hydroxylamine 27: Iodosobenzoic acid
28: Lys-C 29: Lys-N 30: Neutrophil elastase
31: NTCB 32: Papain 33: Pepsin pH 1.3
34: Pepsin pH >=2 35: Proline-endopeptidase 36: Proteinase K
37: Staphylococcal peptidase I 38: Thermolysin 39: Thrombin (PeptideCutter)
40: Thrombin SG 41: Tobacco etch virus protease 42: Trypsin

2.1.1. Arg-C

Arg-C proteinase preferentially cleaves after R (P1)

RPG definition:

cleaving rule:

  • (R,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#ArgC

2.1.2. Asp-N

Asp-N Sequencing Grade preferentially cleaves before C or D (P1’ )

RPG definition:

cleaving rule:

  • (,C or D)

More information: https://france.promega.com/resources/pubhub/using-endoproteinases-asp-n-and-glu-c-to-improve-protein-characterization/

2.1.3. BNPS-Skatole

BNPS-Skatole preferentially cleaves after W (P1)

RPG definition:

cleaving rule:

  • (W,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#BNPS

2.1.4. Bromelain

Bromelain preferentially cleaves after K, A or Y (P1)

RPG definition:

cleaving rule:

  • (K or A or Y,)

More information: https://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=16410479

2.1.5. Caspase 1

Caspase 1 preferentially cleaves after D (P1) preceded by H, A or T in P2 and preceded by F, W, Y or L in P4. It will not cleave if D is followed by P, E, D, Q ,K or R in P1’.

RPG definition:

cleaving rule:

  • (F or W or Y or L)()(H or A or T)(D,)

exception rule:

  • (F or W or Y or L)()(H or A or T)(D,)(P or E or D or Q or K or R)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Casp1

2.1.6. Caspase 2

Caspase 2 preferentially cleaves after D (P1) preceded by DVA or DEH. It will not cleave if D is followed by P, E, D, Q ,K or R in P1’.

RPG definition:

cleaving rules:

  • (D)(V)(A)(D,)
  • (D)(E)(H)(D,)

exception rules:

  • (D)(V)(A)(D,)(P or E or D or Q or K or R)
  • (D)(E)(H)(D,)(P or E or D or Q or K or R)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Casp2

2.1.7. Caspase 3

Caspase 3 preferentially cleaves after D (P1) preceded by DMQ or DEV. It will not cleave if D is followed by P, E, D, Q ,K or R in P1’.

RPG definition:

cleaving rules:

  • (D)(M)(Q)(D,)
  • (D)(E)(V)(D,)

exception rules:

  • (D)(M)(Q)(D,)(P or E or D or Q or K or R)
  • (D)(E)(V)(D,)(P or E or D or Q or K or R)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Casp3

2.1.8. Caspase 4

Caspase 4 preferentially cleaves after D (P1) preceded by LEV or (W/L)EH. It will not cleave if D is followed by P, E, D, Q ,K or R in P1’.

RPG definition:

cleaving rules:

  • (L)(E)(V)(D,)
  • (W or L)(E)(H)(D,)

exception rules:

  • (L)(E)(V)(D,)(P or E or D or Q or K or R)
  • (W or L)(E)(H)(D,)(P or E or D or Q or K or R)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Casp4

2.1.9. Caspase 5

Caspase 5 preferentially cleaves after D (P1) preceded by (W/L)EH.

RPG definition:

cleaving rule:

  • (W or L)(E)(H)(D,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Casp5

2.1.10. Caspase 6

Caspase 6 preferentially cleaves after D (P1) preceded by VEI or VEH. It will not cleave if D is followed by P, E, D, Q ,K or R in P1’.

RPG definition:

cleaving rule:

  • (V)(E)(I or H)(D,)

exception rule:

  • (V)(E)(I or H)(D,)(P or E or D or Q or K or R)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Casp6

2.1.11. Caspase 7

Caspase 7 preferentially cleaves after D (P1) preceded by DEV. It will not cleave if D is followed by P, E, D, Q ,K or R in P1’.

RPG definition:

cleaving rule:

  • (D)(E)(V)(D,)

exception rule:

  • (D)(E)(V)(D,)(P or E or D or Q or K or R)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Casp7

2.1.12. Caspase 8

Caspase 8 preferentially cleaves after D (P1) preceded by (I/L)ET. It will not cleave if D is followed by P, E, D, Q ,K or R in P1’.

RPG definition:

cleaving rule:

  • (I or L)(E)(T)(D,)

exception rule:

  • (I or L)(E)(T)(D,)(P or E or D or Q or K or R)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Casp8

2.1.13. Caspase 9

Caspase 9 preferentially cleaves after D (P1) preceded by LEH.

RPG definition:

cleaving rule:

  • (L)(E)(H)(D,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Casp9

2.1.14. Caspase 10

Caspase 10 preferentially cleaves after D (P1) preceded by IEA.

RPG definition:

cleaving rule:

  • (I)(E)(A)(D,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Casp10

2.1.15. Chymotrypsin high specificity

This chymotrypsin preferentially cleaves after F, Y or W (P1) if not followed by P in P1’. It will not cleave after W followed by M in P1’.

RPG definition:

cleaving rule:

  • (F or Y or W,)

exception rules:

  • (F or Y or W,)(P)
  • (W,)(M)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Chym

2.1.16. Chymotrypsin low specificity

This chymotrypsin preferentially cleaves after F, L, Y, W, M or H (P1) if not followed by P in P1’. It will not cleave after W followed by M in P1’. It will not cleave after M followed by Y in P1’. It will not cleave after H followed by D/M/W in P1’.

RPG definition:

cleaving rule:

  • (F or L or Y or W or M or H,)

exception rules:

  • (F or L or Y or W or M or H,)(P)
  • (W,)(M)
  • (M,)(Y)
  • (H,)(D or M or W)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Chym

2.1.17. Clostripain

Clostripain (Clostridiopeptidase B) preferentially cleaves after R (P1).

RPG definition:

cleaving rule:

  • (R,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Clost

2.1.18. CNBr

CNBr preferentially cleaves after M (P1).

RPG definition:

cleaving rule:

  • (M,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#CNBr

2.1.19. Enterokinase

Enterokinase preferentially cleaves after K (P1) preceded by D/E in P2, P3, P4 and P5.

RPG definition:

cleaving rule:

  • (D or E)(D or E)(D or E)(D or E)(K,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Enter

2.1.20. Factor Xa

Factor Xa preferentially cleaves after R (P1) preceded by G in P2, D/E in P3 and A/F/I/L/V/W/G/T in P4.

RPG definition:

cleaving rule:

  • (A or F or I or L or V or W or G or T)(D or E)(G)(R,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Xa

2.1.21. Ficin

Ficin preferentially cleaves after G, S, E or Y (P1) preceded by A, V, I, L ,F, Y or W in P2.

RPG definition:

cleaving rule:

  • (A or V or I or L or F or Y or W)(G or S or E or Y,)

More information: https://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=16410578

2.1.22. Formic acid

Formic acid preferentially cleaves after D (P1).

RPG definition:

cleaving rule:

  • (D,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#HCOOH

2.1.23. Glu-C

Glu-C Sequencing Grade preferentially cleaves after D or E (P1).

RPG definition:

cleaving rule:

  • (D or E,)

More information: https://france.promega.com/resources/pubhub/using-endoproteinases-asp-n-and-glu-c-to-improve-protein-characterization/

2.1.24. Glutamyl endopeptidase

Glutamyl endopeptidase preferentially cleaves after E (P1).

RPG definition:

cleaving rule:

  • (E,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Glu

2.1.25. Granzyme B

Granzyme B preferentially cleaves after D (P1) preceded by IEP.

RPG definition:

cleaving rule:

  • (I)(E)(P)(D,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#GranB

2.1.26. Hydroxylamine

Hydroxylamine (NH2OH) preferentially cleaves after N (P1) followed by G in P1’.

RPG definition:

cleaving rule:

  • (N,)(G)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Hydro

2.1.27. Iodosobenzoic acid

Iodosobenzoic acid preferentially cleaves after W (P1).

RPG definition:

cleaving rule:

  • (W,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Iodo

2.1.28. Lys-C

LysC Lysyl endopeptidase (Achromobacter proteinase I) preferentially cleaves after K (P1).

RPG definition:

cleaving rule:

  • (K,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#LysC

2.1.29. Lys-N

LysN Peptidyl-Lys metalloendopeptidase preferentially cleaves before K (P1’ ).

RPG definition:

cleaving rule:

  • (,K)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#LysN

2.1.30. Neutrophil elastase

Neutrophil elastase preferentially cleaves after A or V (P1).

RPG definition:

cleaving rule:

  • (A or V,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Elast

2.1.31. NTCB

NTCB +Ni (2-nitro-5-thiocyanobenzoic acid) preferentially cleaves before C (P1’ ).

RPG definition:

cleaving rule:

  • (,C)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#NTCB

2.1.32. Papain

Papain preferentially cleaves after R or K (P1) preceded by A, V, I, L ,F, Y or W in P2. It will not cleave if followed by V in P1’.

RPG definition:

cleaving rule:

  • (A or V or I or L or F or Y or W)(R or K,)

exception rule:

  • (A or V or I or L or F or Y or W)(R or K,)(V)

More information: https://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=16410606

2.1.33. Pepsin pH 1.3

This pepsin preferentially cleaves around F or L (P1 or P1’ ). It will not cleave before F or L in P1’ followed by P in P2’. It will not cleave before F or L in P1’ preceded by R in P1 or P in P2 or H/K/R in P3. It will not cleave after F or L in P1 followed by P in P2’. It will not cleave after F or L in P1 preceded by P in P2 or H/K/R in P3.

RPG definition:

cleaving rule:

  • (,F or L,)

exception rules:

  • (,F or L)(P)
  • (R)(,F or L)
  • (P)()(,F or L)
  • (H or K or R)()()(,F or L)
  • (F or L,)()(P)
  • (P)(F or L,)
  • (H or K or R)()(F or L,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Peps

2.1.34. Pepsin pH >=2

This pepsin preferentially cleaves around F, L, W or Y (P1 or P1’ ). It will not cleave before F, L, W or Y in P1’ followed by P in P2’. It will not cleave before F, L, W or Y in P1’ preceded by R in P1 or P in P2 or H/K/R in P3. It will not cleave after F, L, W or Y IN P1 followed by P in P2’. It will not cleave after F, L, W or Y in P1 preceded by P in P2 or H/K/R in P3.

RPG definition:

cleaving rule:

  • (,F or L or W or Y,)

exception rules:

  • (,F or L or W or Y)(P)
  • (R)(,F or L or W or Y)
  • (P)()(,F or L or W or Y)
  • (H or K or R)()()(,F or L or W or Y)
  • (F or L or W or Y,)()(P)
  • (P)(F or L or W or Y,)
  • (H or K or R)()(F or L or W or Y,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Peps

2.1.35. Proline-endopeptidase

Proline-endopeptidase preferentially cleaves after P (P1) preceded by H, K or R in P2 but will not cleaves if followed by P in P1’.

RPG definition:

cleaving rule:

  • (H or K or R)(P,)

exception rule:

  • (H or K or R)(P,)(P)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Pro

2.1.36. Proteinase K

Proteinase K preferentially cleaves after F, W, Y, T, E, A, V, L or I (P1). The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids.

RPG definition:

cleaving rule:

  • (F or W or Y or T or E or A or V or L or I,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#ProtK

2.1.37. Staphylococcal peptidase I

Staphylococcal peptidase I preferentially cleaves after E (P1). It will not cleave after E in P1 preceded by E in P2, but cleaves after E in P1 followed by E in P1’.

RPG definition:

cleaving rule:

  • (E,)

exception rule:

  • (E)(E,)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Staph

2.1.38. Thermolysin

Thermolysin preferentially cleaves before A,F,I,L,M or V (P1’ ) when not followed by P in P2’ nor preceded by D or E in P1.

RPG definition:

cleaving rule:

  • (,A or F or I or L or M or V)

exception rules:

  • (,A or F or I or L or M or V)(P)
  • (D or E)(,A or F or I or L or M or V)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Therm

2.1.39. Thrombin (PeptideCutter)

This thrombin preferentially cleaves after R (P1). Optimum cleavage is when R is preceded and followed by G (P2 and P1’ ). Cleavage also occurs when R is preceded by P in P2 and A, F, I, L, V, W, G or T in P3 and P4. It will not cleave after R followed by D/E in P1’ or P2’.

It is not strictly coherent with the definition in PeptideCutter, as in this software there are differences between definition, summary and behavior of this enzyme.

RPG definition:

cleaving rules:

  • (G)(R,)(G)
  • (A or F or I or L or V or W or G or T)(A or F or I or L or V or W or G or T)(P)(R,)

exception rules:

  • (A or F or I or L or V or W or G or T)(A or F or I or L or V or W or G or T)(P)(R,)(D or E)
  • (A or F or I or L or V or W or G or T)(A or F or I or L or V or W or G or T)(P)(R,)()(D or E)

Warning

the following combined exception (A or F or I or L or V or W or G or T)(A or F or I or L or V or W or G or T)(P)(R,)(D or E)(D or E) cannot be used instead, as it will cleave on […](R,)(D or E).

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Throm https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3288055/

2.1.40. Thrombin SG

This thrombin (Sequencing Grade) preferentially cleaves after R (P1) preceded by P in P2, V in P3 and L in P4 and followed by G in P1’ and S in P2’.

This thrombin is defined in several kits (see below).

RPG definition:

cleaving rule:

  • (L)(V)(P)(R,)(G)(S)

More information: see thrombin cleavage kits of Abcam, BioVision, Merck or Novagen.

2.1.41. Tobacco etch virus protease

Tobacco etch virus protease (TEV) preferentially cleaves after Q (P1) when followed by G or S in P1’ and preceded by Y in P3 and E in P6.

RPG definition:

cleaving rule:

  • (E)()()(Y)()(Q,)(G or S)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#TEV

2.1.42. Trypsin

Trypsin preferentially cleaves after K or R (P1). It will not cleave after K followed by P in P1’ except if W in P2. It will not cleave after R followed by P in P1’ except if M in P2. It will not cleave CKD, DKD, CKH, CKY, CRK, RRH nor RRR.

RPG definition:

cleaving rules:

  • (K or R,)
  • (W)(K,)(P)
  • (M)(R,)(P)

exception rules:

  • (K or R,)(P)
  • (C)(K,)(D)
  • (D)(K,)(D)
  • (C)(K,)(H)
  • (C)(K,)(Y)
  • (C)(R,)(K)
  • (R)(R,)(H)
  • (R)(R,)(R)

More information: https://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html#Tryps