(1) The UniRef50 entry might include paralogous sequences which although at least 50% identical to the sequence with the known cleavage, might be processed or degraded differently and there is no evolutionary pressure to maintain the known cleavage site. Where a cleavage site was not conserved, a paralogue was identified in an alignment as a second protein from the same species that was clearly not a splice variant. (2) UniRef50 entries contain many translated genes from genome sequencing projects; gene finding in eukaryote genomes is notoriously difficult and it is possible that erroneous gene building has resulted, for example, in the loss of the exon encoding the cleavage site or the inclusion of part of an intron in its place. (3) It is also probable that for some peptidases there are not enough substrates known to be sure that any amino acid is really excluded from a particular binding site. The number of substrates known for each peptidase is included in Table 4 , because the greater the number of substrates the more likely that an amino acid is really atypical and not just unobserved. (4) The alignment is incorrect. This is unlikely given the close relationship between the sequences, which are all 50% or more identical; however there are situations where an insert or deletion occurs within the range P4-P4 0 . (5) Some endogenous cleavages (for example removal of signal and transit peptides) may be the result of more than one cleavage, because aminopeptidases nibble away the N-terminus (1), and may thus be incorrectly mapped to the specificity of the leader peptidase. (6) It is theoretically possible that if the substrate and peptidase are from the same organism both will have evolved to accommodate a change in the cleavage position. (7) A single residue mismatch may also be due to a single-base sequencing error. Potential errors of this kind can be identified using a codon dictionary, provided the atypical residue could be the result of a single base change, and that it is the only residue not conserved, regardless of the number of sequences in the alignment. (8) Some cleavages regarded as 'physiological' are actually fortuitous. If a cleavage site is extremely poorly conserved it is unlikely to be physiologically relevant. 