Query Prohormone Database

The PepShop database can be searched by UniProt accession number, gene symbol, organism name (7 species), exact amino acid sequence, and peptide monoisotopic mass with adjustable mass tolerance level.

1. Accession Number

The primary accession numbers in one of the following databases: UniProt (Examples: P22005, P01193), NCBI-RefSeq (Examples: NP_079142.2, NP_891558.1), UniGene (Examples: Mm.210541, Rn.141298, Hs.1897), Mouse genome index database (Examples: MGI:108058, MGI:2675256), Entrez Gene (Examples: 223780, 4879, 24602).

2. Prohormone Symbol

The prohormone symbol (Examples: "ADML", "COLI").

3. Species

PepShop supports the prohormone and peptide identification based on the availability of sequenced genomes. Currently information is available for the Human, Mouse, Rat, Rhesus monkey, Cattle, Dog and Pig species. User can select the organism name from the pull down menu.

4. Monoisotopic Mass

The monoisotpic mass of peptide with adjustable mass tolerance (±).

5. Prohormone Sequence

The full or partial exact prohormone or peptide amino acid sequence. There are two supported sequence formats: FASTA format or only sequence without FASTA header (Examples: "YGGFM", "KYVMGHFRWD"). Multiple sequence submissions are not supported at the same time.

Bioinformatic Tools

Bioinformatics tools for prediction of cleavage sites in prohromone sequences, single sequence search (BLASTP) and Multiple Sequence Alignment (MUSCLE) have been provided in PepShop.

The basic use of these tools requires:

2. NeuroPred

a). Model Selection

The following cleavage prediction models are available, the first model is an empirical model, whereas all other models are binary logistic regression models.

The following models are available:

Known Motif:

This model, reported by Southey et al. (2006a), is based on the occurrence of the following motifs in the sequence: xxKR, xxKK, xxRR, RxxR and RxxK, where x is any amino acid and cleavage occurs after the right-most or C-terminal amino acid. When any of these motifs is present, the cleavage probability is set to 0.88 and when two of these motifs are present (e.g. RxKR, which is composed of the motifs xxKR and RxxR), the cleavage probability is set to 0.997. This is the default model.

Mollusc:

The Complex Model, reported by Hummon et al. (2003), trained on precursor sequences from the mollusc, Aplysia californica.

Mammalian:

Model reported by Amare et al. (2006), trained on published mammalian precursor sequences from cow, human, mouse, pig and rat.

Insect:

A joint model derived from the models trained by Southey et al. (2008) on published precursor sequences from the honey bee, Apis mellifera and fruit fly, Drosophila melanogaster.

Mollusc_Basic:

Model reported by Hummon et al. (2003), trained on precursor sequences from the mollusc, Aplysia californica. This model has fewer terms than the Mollusc model and also had poorer predictive ability than the Mollusc mode (Hummon et al. 2003).

Apis:

Model reported by Southey et al. (2008), trained on published precursor sequences from the honey bee, Apis mellifera, reported by Hummon et al., 2006.

Drosophila:

Model reported by Southey et al. (2008), trained on precursor sequences from the fruit fly, Drosophila melanogaster

Bovine:

A model trained on published cattle, Bos taurus, precursor sequences by Tegge et al. (2007, 2008).

Human:

A model trained on published human, Homo sapiens, precursor sequences by Tegge et al. (2007, 2008).

Mouse:

A model trained on published mouse, Mus musculus, precursor sequences by Tegge et al. (2007, 2008).

Rat:

A model trained on published rat, Rattus norvegicus, precursor sequences by Tegge et al. (2007, 2008).

Any Basic Site:

Simply considers any basic amino acid (Arginine or Lysine) as cleaved. However, when the amino acid combinations listed in the known motif model (e.g. xxKR, xxKK, xxRR, RxxR and RxxK) are present, only the last amino acid in the motif is considered as cleaved.

b). Selection of Post-Translational Modifications

Prohormone precursors undergo extensive modification before active neuropeptides and hormones are obtained. In addition to cleavages at basic sites and immediate removal of C-terminal basic residues (Trim C-terminal K and R), several other modifications may be present. The most common PTMs are amidation (of C-terminal glycine) and pyroglutamylation (cyclization of N-terminal glutamate or glutamine). Sulfation of tyrosine (Tyr-Sulfation) and acetylation are also common, albeit occurring somewhat less frequently; thus these four common PTMs are grouped together while even less common PTMs are grouped separately. Disulfide bond formation between two cysteine residues resulting in a mass loss of 2 Da is a common PTM in neuropeptides, but it is difficult to predict whether the disulfide bond is formed between two peptides or within a peptide containing two or more cysteines. In addition, it is difficult to identify the cysteine pairs involved in bond formation. For these reasons, disulfide bond formation is not modeled in NeuroPred. The user may consult web-based tools such as Cyspred to determine potentially disulfide bonding cysteines. A table of the available PTMs can be found here.

Post-Translational Modifications (PTMs)

Trim C-terminal K and R
Most Common PTMs	Less Common PTMs
Amidation	O-linked Glycosylation of S	N-linked Glycosylation of S	Bromination of W
Pyroglutamination	O-linked Glycosylation of T	N-linked Glycosylation of T	Methylation of E
Acetylation	Dipeptidase	Hydroxylation of P	Methylation of H
Sulfation of Y	Carboxylation of E	Phosphorylation of S	Methylation of K
	DiAcetylation	Phosphorylation of T	Methylation of R

3. Blastp

Standard protein-protein BLAST (blastp) will attempt to identify both submitted amino acid sequences and other similar sequences in selected protein database. The blastp find local regions of similarity using pairwise sequence alignment. When sequence similarity spans the whole sequence, blastp will also report a global alignment, which is the preferred result for prohormone identification purposes. The following databases are available in PepShop for blastp: RefSeq-mammalian, RefSeq-invertebrate, RefSeq-othervertebrates, UniProt and PepShop prohormone databases.

4. Muscle

Multiple Sequence Alignment (MSA) of two or more than two submitted sequences can be performed using Muscle. To get alignment, click on

D. References

Amare, A., Hummon, A.B., Southey, B.R., Zimmerman, T.A., Rodriguez-Zas, S.L., Sweedler, J.V., Bridging neuropeptidomics and genomics with bioinformatics: prediction of mammalian neuropeptide prohormone processing. J. Proteome Res. 2006, 5, 1162-1167. Abstract.

Hummon, A.B., Hummon, N.P., Corbin, R.W., Li, L.J., Vilim, F.S., Weiss, K.R., Sweedler, J.V., From precursor to final peptides: a statistical sequence-based approach to predicting prohormone processing. J. Proteome Res. 2003, 2, 650-656. Abstract.

Hummon, A.B. Richmond, T.A. Verleyen, P. Baggerman, G. Huybrechts, J. Ewing, M A. Vierstraete, E. Rodriguez-Zas, S.L. Schoofs, L. Robinson, G.E. Sweedler, J.V. , From the Genome to the Proteome: Uncovering Peptides in the Apis Brain, Science 2006, 314, 647-649. Abstract.

Southey, B.R., Rodriguez-Zas, S.L., Sweedler, J.V., Prediction of neuropeptide prohormone cleavages with application to RFamides. Peptides 2006a, 27, 1087-1098. Abstract.

Southey B.R., Amare A., Zimmerman T.A., Rodriguez-Zas S.L., Sweedler J.V., NeuroPred: a tool to predict cleavage sites in neuropeptide precursors and provide the masses of the resulting peptides. Nucleic Acids Res. 2006b, 34 (Web Server issue), W267-272. Abstract.

Tegge, A.N. Southey, B.R. Sweedler, J.V. Rodriguez-Zas, S.L., Enhanced Prediction of Cleavage in Bovine Precursor Sequences. Lecture Notes in Computer Science, Bioinformatics Research and Applications, Vol. 4463, pp. 350-360, 2007, Springer. Abstract.

Southey, B.R., Hummon, A.B., Richmond, T.A., Sweedler, J.V., Rodriguez-Zas, S.L., Prediction of neuropeptide cleavage sites in insects. Bioinformatics, 2008, 24, 815-825. Full Text

Tegge, A.N., Southey, B.R., Sweedler, J.V., Rodriguez-Zas, S.L., Comparative Analysis of Neuropeptide Cleavage Sites in Human, Mouse, Rat, and Cattle. Mamm. Genome, 2008 , 19(2), 106-120. Abstract.

Questions or comments: Sandra Rodriguez Zas (rodrgzzs@illinois.edu)