Peptide mass calculator

How to calculate a protein digest's peptide masses

Paste a protein sequence or FASTA records, choose an enzyme, and click Calculate. ProteinIQ predicts peptide sequences, their positions, and neutral masses or ion mass-to-charge ratios (m/z). It runs in the browser and supports fixed cysteine modifications, methionine oxidation, mass-window filtering, and missed cleavages.

Trypsin example

The Trypsin digest example uses this input:

>Trypsin_example
AKR

With the default settings, the results are:

These values are m/z for singly protonated [M+H]+ ions. Setting Missed cleavages to 1 also includes AKR, at positions 1-3, with m/z 374.2510 and one missed cleavage. Fully cleaved peptides remain in the results.

Multiple proteins

Each protein needs its own header. The Two proteins example contains:

>First
AK
>Second
GK

The default results are AK at positions 1-2, m/z 218.1499, for First, and GK at positions 1-2, m/z 204.1343, for Second.

Multiply charged ions

For the raw sequence PEPTIDE, select [M+2H]²⁺ and keep the other defaults. The peptide spans positions 1-7; its reported m/z is 400.6873. Its neutral mass is 799.3599 Da. The selected ion type changes the quantity being reported and filtered.

Input

Give every protein a unique FASTA identifier. For a UniProt header such as sp|P01308|INS_HUMAN, the results use accession P01308. Repeated identifiers produce a warning. A raw input is one sequence named Sequence; separate raw lines are joined, so a list of proteins needs FASTA headers.

Empty records, alignment gaps, stop markers, unsupported ambiguity codes (B, Z, X, J), and inline modifications fail the calculation instead of being deleted. Put names such as GFP in a >GFP header: names made entirely of valid amino acid letters cannot be distinguished from raw sequence data. For spreadsheet columns, use CSV to FASTA first. Word and PDF files are not accepted directly; paste their sequence text.

Settings

Both bounds must be finite, non-negative numbers. Filtering uses the calculated value before four-decimal rounding. If nothing falls within the window, widen the bounds or check the selected ion type.

Enzyme selection

These are the calculator's current simplified cleavage rules, also used for cleavage-site analysis. They predict positions rather than digestion efficiency.

The Glu-C buffer distinction follows ExPASy's documented specificity: phosphate cleaves D/E, while bicarbonate cleaves E. These simplified rules omit ExPASy's adjacent-residue exceptions for Glu-C and some context exceptions for other enzymes. Verify the actual rule above before interpreting an experimental digest.

Results

Use the results table's copy and export controls to reuse the peptide list. Two fragments can have the same mass; positions and identifiers distinguish them. Warnings identify repeated protein IDs and the unmodeled chemistry of CNBr cleavage.

How peptide mass is calculated

The calculator sums residue masses, adds one water molecule for free termini, and applies fixed modification increments. Residue values follow the ExPASy mass table, including pyrrolysine. Water contributes 18.01056 Da in monoisotopic mode or 18.01528 Da in average mode. The proton mass used for ion calculations is 1.00728 Da.

For neutral mass $M$ and positive charge $z$:

For [M-H]⁻, the reported m/z is $M-m_p$. Monoisotopic masses use the dominant isotope of each element; average masses use natural-isotope averages. The constants and four-decimal display precision make these predictions suitable for planning and comparison, not instrument calibration.

Limitations

• Fixed modifications affect all matching residues. The calculator does not enumerate variable oxidation or alkylation combinations, disulfide bonds, phosphorylation, glycosylation, terminal amidation, or cyclic peptides.
• CNBr cleavage converts methionine into homoserine products experimentally. This calculator retains methionine mass and ordinary free termini, and shows a warning. Its CNBr results are a cleavage-position model, not chemically corrected product masses.
• Signal peptides, mature chains, UniProt annotations, and structure accessibility are not automatically modeled. The entered sequence is digested in full.
• Cleavage is deterministic. Missed cleavages enumerate possibilities without assigning probabilities or predicting which peptides an instrument will detect.

Which tool should I use?

FAQ

Why does a doubly charged peptide report roughly half its neutral mass?

The result is m/z. Two protons are added to the neutral mass, then the total is divided by charge 2. Select [M] (neutral) for molecular mass in Da.

Can I calculate the mass of a peptide without breaking it into fragments?

This tool always applies the selected cleavage rule. A peptide stays intact only when it has no internal cut sites for that enzyme. It has no no-digestion setting.

Why are ambiguous residues rejected?

B, Z, and X do not specify a unique residue mass. J is also unsupported here. Replace ambiguity codes with the known residues before comparison; they are never silently removed.