Prediction of Protein Cut Sites with PeptideCutter Web Tool

The PeptideCutter web tool helps predict the cleavage sites of proteases or various chemicals in protein sequences. Protease cutting can be useful for experimenting on a specific part of the protein, isolating domains in the protein, removing the tag protein when expressing a fusion protein, or verifying that the protein under investigation is resistant to endogenous proteases. One or more reagents can be selected from the list of proteases and chemicals when performing the analysis. The query can be entered as the protein sequence, Swiss-Prot/TrEMBL accession number, raw sequence, or a string in FASTA format, in a single-letter amino acid code. Letters that do not correspond to an amino acid code (B, J, O, U, X, or Z) will result in an error message and the user must correct the query (1). Different output formats are offered for the results. In these outputs, the cut sites can be displayed either alphabetically by enzyme name or in a table sorted by amino acid number. A third output option is a map of the cut sites. The sequence itself and the cut regions marked on it are grouped according to the block sizes specified by the user, providing an easy printing format.

In the example application, the Hemoglobin Subunit Beta (HBB) protein will be used as input in the PeptideCutter web tool.

Step 1: To obtain the FASTA format of the Hemoglobin Subunit Beta (HBB) protein, go to the UniProt database. https://www.uniprot.org/ is used to access UniProt.

Figure 1. Preliminary information for HBB protein in the UniProt database.

Step 2: The FASTA format of the structure coded P68871 for the HBB protein of Homo sapiens is downloaded from the Download tab on the UniProt page or can be previewed without downloading the file using the Preview option in the tab.

Figure 2. UniProt file download page.

Step 3: The FASTA sequence obtained for the HBB protein is given as input to the PeptideCutter web tool. The PeptideCutter web tool can be accessed at https://web.expasy.org/peptide_cutter/ .

Figure 3. Home page of the PeptideCutter web tool.

Step 4: After the FASTA sequence is added to the input section, analysis can be performed by selecting all cutting enzymes or a specific cutting enzyme from the list if desired. The FASTA sequence of HBB has been entered in the figure below and all cutting enzymes have been selected for analysis (Figure 4). Other settings can be left as default or adjusted as desired.

Figure 4. Input page of the PeptideCutter web tool.

Step 5: After these operations, the analysis is started from the Perform option at the bottom of the page. Since it is a short process, the results are also given on the same screen. It is possible to see the results both in the table and in the map via the series.

Figure 5. Results page of the PeptideCutter web tool.

Step 6: When the results are examined, it is seen that 20 different cutting enzymes and chemicals are involved in the HBB protein and at which positions these enzymes cut the HBB protein. For example, it was determined that the trypsin enzyme has a cutting site at amino acids at positions 9, 18, 31, 41, 60, 62, 66, 67, 83, 96, 105, 121, 133 and 145 on the HBB protein (Figure 5). In addition, it is seen that 17 cutting enzymes do not have a cutting site on the HBB protein (Figure 6).

Figure 6. Enzymes without a cutting site on the HBB protein.

Step 7: The areas where the cutting enzymes will cut on the HBB protein sequence can also be seen as a map.

Figure 7. Mapping of the cut sites on the sequence of the HBB protein (between the 121st and 147th amino acids).

PeptideCutter web tool is a powerful tool for fast and accurate analysis of protease cleavage sites in protein sequences. The tool allows users to work on both database and manually entered protein sequences and provides comprehensive results with different protease or chemical options. Visualization of cleavage sites in table or map form makes it easier to understand analysis results. Especially for researchers conducting experimental studies on protein structure and function, PeptideCutter web tool provides an effective planning and evaluation process.

REFERENCES

1. Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A. (2005). Protein Identification and Analysis Tools on the Expasy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press.