Peptide cutter

How to find protease cleavage sites online

Peptide Cutter maps potential cleavage sites in one or more proteins using 41 protease and chemical cleavage rules. Paste a sequence or upload protein FASTA, choose the enzymes, and click Analyze. The calculation runs in the browser.

1. Put each protein below a separate FASTA header, or paste a single unnamed sequence.
2. Keep All enzymes (41) for a survey, or choose Custom selection to inspect particular enzymes.
3. Leave both cut-count filters blank to include every predicted site.
4. Read Position, Cleave side, and Context window together to locate the bond. Use Download to save the displayed results as CSV.

Example: a TEV tag-removal site

Select only TEV protease under Custom selection and analyze this linker:

>tag_linker
ENLYFQG

The result is one row:

The pipe marks the bond after Q6. Leading dots mean the excerpt starts partway through the input, not that residues are missing from the calculation.

Example: multiple FASTA records and N-terminal cuts

Select only Trypsin and Asp-N:

>sp|P12345|A
AKR
>tr|Q12345|B
ADDA

With the default position sort, these synthetic records produce:

For an N-terminal cut, Position identifies the residue after the bond. Asp-N at position 2 means cleavage before D2. UniProt headers use the accession field, so these records remain distinguishable.

Input

Alignment gaps, stop markers, ambiguous residues (B, Z, X, J), modification annotations, and digits inside sequence blocks are rejected. They are never silently removed. Empty FASTA records also stop the run so a protein cannot disappear unnoticed.

Put names and descriptions on > header lines. Words made entirely of amino-acid letters, such as GFP, cannot be distinguished from a sequence when pasted without a header. Compact >name SEQUENCE records remain accepted when there are no sequence lines, with a warning about that interpretation. Conventional FASTA with the sequence on the next line avoids this ambiguity.

GenBank, FASTQ, PDB, spreadsheet, and alignment text must be converted to protein FASTA first. Use FASTA converter for sequence text or PDB to FASTA for a structure file.

Settings

Results

Each row represents one potential cleavage event under a selected sequence rule. Cut-count filters apply separately to each enzyme in each protein and include their boundary values.

A valid protein with no predicted sites returns an empty result with a notice. A separate notice explains when cut-count filters hide all sites for a protein. In a multi-record run, proteins without displayed sites do not contribute rows, and the notice reports how many were affected.

Supported enzymes

These tables describe the 41 implemented rules, including specificity variants. They are a compact sequence-rule model rather than an exact reproduction of every condition and exception in the ExPASy PeptideCutter specificity reference.

Serine proteases

Cysteine proteases

All caspases cleave after the terminal Asp and are blocked by Pro at P1'.

Aspartyl proteases

Metalloproteases

Endopeptidases

Chemical reagents

How Peptide Cutter works

Each enzyme uses a primary-sequence rule. Simple rules match one residue and optional neighboring exclusions; motif rules recognize several residues; context rules check alternative local patterns. The residue positions around a cut are often described as P4-P3-P2-P1 | P1'-P2'-P3'-P4'.

Only internal peptide bonds are reported. A C-terminal matching residue at the end creates no cut, and an N-terminal matching residue at the start creates no cut.

The Glu-C buffer distinction follows ExPASy PeptideMass Table 1: phosphate-buffer Glu-C cleaves after D or E, while bicarbonate-buffer Glu-C cleaves after E. For ADEA, selecting both variants gives phosphate cuts after D2 and E3, and a bicarbonate cut after E3.

Which peptide tool should I use?

Limitations

Specificity rules use primary sequence only. Structure, accessibility, modifications, enzyme source, reaction conditions, and digestion time can change experimental cleavage. A listed site is a candidate for cleavage, not a probability, a guaranteed product, or an upper bound on all possible cuts.

Several rules use simplified residue sets or motifs. Trypsin uses the K/R rule with a following-proline block, without ExPASy's extended contextual exceptions. Broad proteases and caspase motifs do not cover all reported substrate preferences. The local Glu-C rules do not model the additional adjacent-residue exceptions in the ExPASy table. Review the implemented rules and validate important sites experimentally.

FAQ

Does Peptide Cutter calculate fragment masses?

It reports sites, not fragment masses or missed-cleavage products. Use the peptide mass calculator for those results, or FindPept when matching experimental masses to a sequence.

Why is the same position listed several times?

Different enzymes can recognize the same bond. Each enzyme has its own row, even when its cleavage site overlaps another enzyme's site.

Why are there no results for my protein?

The selected rules may have no matches, or the cut-count filters may exclude every matching enzyme. The notice distinguishes these cases. Clear the filters or select more enzymes to inspect the alternatives.