In SWISS-PROT sequences with 'SIGNAL' in the FT line were extracted.
Only sequence entries with experimental evidence were used and signal
peptide entries marked 'HYPOTHETICAL', 'POTENTIAL', 'PROBABLE' were
removed. Furthermore were entires with more than one cleavage site
From secretory proteins, the sequence of the signal peptide and the
first 30 amino acids of the mature protein were included in the data
set. From cytoplasmic and (for the eukaryotes) nuclear proteins, the
first 70 amino acids of each sequence were used. Additionally, a set
of eukaryotic signal anchor sequences, i.e. N-terminal parts
of type II membrane proteins, were extracted.
Redundancy in the data sets was avoided by excluding pairs of sequences
which were functionally homologous, i.e. where the cleavage
site of one signal peptide could be located by simply aligning it to
the other. The numbers of sequences remaining in the
final data sets (version 3) are shown below:
||Number of sequences
| Signal peptides
|| Cytoplasmic proteins
|| Signal anchors
|| Nuclear proteins
|| 1192 || 459 || 67 || 990
|| 358 || 334 || - || -
|| 153 || 151 || - || -
The redundancy reduction is described in detail in
"Defining a similarity threshold for a functional protein sequence
pattern: The signal peptide cleavage site",
Henrik Nielsen, Jacob Engelbrecht, Gunnar von Heijne, and
Proteins, 24, 165-177, 1996.
The abstract can be found here.
Data set cleanup
We were able to identify wrongly annotated signal peptides from
Swiss-Prot simply by calculating the isoeletric point of the
signal peptide and corresponding mature protein.
Using this approach we were even able to identify wrongly
annotated start codons. Indepth information on this issue can be
found in "Improved prediction of signal peptides - SignalP 3.0 ", Bendtsen et al., 2004. The abstract can
be found here..
In Swiss-Prot, annotated signal peptide cleavage sites are entries found do in some
cases contain propeptides. These sequence entries were
reannotated acording to the output of SignalP version 2.
Propeptides have basic residues (R/K) at their cleavage sites.
Spurious residues at position -1 was also removed in the training
set. Only amino acids A,L,G,xxx were alowed at this position.
Wrong start codons
In specific cases, SignalP can be used as a "validation" tool for correct startcodon annotation. In
Swiss-Prot entry SFMA_ECOLI (Swiss-Prot rel. 40) the annotated Signal peptide cleavage site was
positioned 22 and a predicted cleavage site at position at 34. As the S-score (indicating signal
peptide-ness) is very low until position 11 we believed that the methionine at position 12 is the true
start of the protein. In a later release of Swiss-Prot this was indeed verified.