Enzyme cleavage rules and PeptideCutter at ExPasy

Question

[Pfs]

I have a question regarding the cleavage rules interpretation in PeptideCutter. I contacted the helpdesk but got no reply.

How are the cleavage patterns interpreted at the end of the sequences? Towards the 3' end of a protein sequence, when the segment to consider is shorter than the actual pattern, are positions P1' and P2' (after the cut) ignored?

For example, consider the case of THROMBIN. When seq = AAPR, 1 cleavage site is reported at position 4. This makes me believe that P1' and P2' are ignored because the sequence is shorter than the pattern.

However, when seq = AAGR, no cuts are reported, which makes me believe P1' (and P2') are NOT ignored.

Answer 1

[Elisabeth Gasteiger]

Apologies for the late reply to your helpdesk query!

If a sequence is shorter than the 8 amino acids under consideration (three positions downstream and 4 positions upstream of each investigated position), or if a potential cleavage site is encountered at or near the termini, the input sequence is completed with "X" residues which cause it to match any amino acid.

This results in a different behaviour for "positive" conditions such as "P1' must be a G" and for "negative" conditions such as "P1' must not be D or E" (as in the 2 cleavage rules for Thrombin), and explains what you observed.

I agree that this may seem slightly problematic. Does it cause problems for your use case? Which is the behaviour you (or others) would expect?