Peptide Calculator

How to Use the Peptide Calculator

1. Enter your peptide sequence using standard single-letter amino acid codes (e.g. ACDEFGHIKLM). FASTA headers and whitespace are ignored automatically.
2. Select N-terminus and C-terminus modifications if applicable (e.g. acetylation, amidation) — these are common in synthetic peptides.
3. Click Calculate Peptide Properties.
4. Review the results: MW in Da and kDa, length, isoelectric point (pI), net charge at pH 7.4, GRAVY hydrophobicity index, extinction coefficient at 280 nm, and full amino acid composition.
5. Use the Charge at a Specific pH field to look up net charge at any pH relevant to your buffer or assay.

The Formulas Behind the Results

Molecular weight is calculated by summing the average residue mass of every amino acid in the sequence, then adding one water molecule (18.015 Da) to account for the free termini. Selecting N-acetylation adds 42.011 Da, and C-amidation subtracts 0.984 Da, reflecting the actual mass change of those chemical modifications. Isoelectric point and net charge both use the Henderson–Hasselbalch equation: for each ionizable group, the fractional positive or negative charge at a given pH is calculated from its pKa, and these are summed across the whole sequence — the pI is the pH where that sum crosses zero. The GRAVY index averages the Kyte–Doolittle hydropathy value of every residue, and the extinction coefficient at 280 nm sums the contribution of Trp (5500 M⁻¹cm⁻¹) and Tyr (1490 M⁻¹cm⁻¹) residues following the Pace et al. 1995 method.

When to Use This Calculator

This tool is most useful right before and after peptide synthesis: use it beforehand to predict the expected molecular weight, charge state, and solubility behavior of a designed sequence, and use it afterward to sanity-check a mass spectrometry result against the theoretical mass. It's also valuable when comparing capped versus uncapped versions of the same peptide, when choosing a buffer pH for storage or chromatography based on the predicted net charge, and when screening candidate peptide sequences for likely solubility problems using the GRAVY score before committing to synthesis.

Common Mistakes to Avoid

• Leaving termini at their default "free" setting for a capped peptide: most synthetic peptides ordered from a vendor are acetylated, amidated, or both — check your certificate of analysis and select the correct modification, or the MW, charge, and pI will all be off.
• Ignoring invalid characters silently: numbers, FASTA headers, or non-standard residue codes pasted into the sequence box are dropped from the calculation; always check the result note for a list of ignored characters.
• Reading GRAVY as a precise solubility prediction: it is a useful screening heuristic based on average hydropathy, not a guarantee — charge distribution and specific motifs can make a peptide behave differently than its GRAVY score alone would suggest.
• Assuming extinction coefficient of zero means no peptide is present: a zero or very low ε₂₈₀ simply means the peptide lacks Trp/Tyr residues; you'll need an alternative quantification method such as BCA or Bradford instead of UV absorbance.

Interpreting Your Results

The molecular weight is your expected target mass for comparison against mass spectrometry. The pI and net charge at pH 7.4 together tell you how the peptide will behave electrically in near-physiological buffer — useful for predicting binding to ion-exchange resin or migration on a native gel. The GRAVY index flags likely solubility behavior: strongly positive values suggest hydrophobic, potentially poorly soluble peptides, while negative values suggest good aqueous solubility. The extinction coefficient lets you convert a measured A280 absorbance reading into peptide concentration via the Beer-Lambert law, provided the peptide contains Trp and/or Tyr. The amino acid composition table breaks down exactly how much each residue contributes to the total mass, which is useful for spotting synthesis errors.

N-terminus and C-terminus Modifications

• Free NH₂ / COOH (default): Unmodified termini — the standard form for most recombinant proteins and natural peptides.
• N-acetylation (+42.011 Da): Common modification that neutralises the positive charge of the N-terminus, increasing metabolic stability. Used in many therapeutic peptides.
• C-amidation (−0.984 Da): Replaces the C-terminal carboxyl group with an amide, neutralising the negative charge and improving membrane permeability and protease resistance.
• Many bioactive peptides (e.g. oxytocin, vasopressin) are C-terminally amidated in vivo.

Frequently Asked Questions

Why do I need to specify N-terminus and C-terminus modifications?

Synthetic peptides are very often capped at one or both ends — N-acetylation neutralises the N-terminal amine's positive charge, while C-amidation removes the C-terminal carboxyl's negative charge. Leaving the default free termini selected when your peptide is actually capped will shift the calculated molecular weight, net charge, and pI away from the true values, sometimes by a meaningful margin for short peptides where the termini make up a larger share of the total charge. Always check your synthesis or vendor certificate of analysis to confirm which modifications, if any, were applied before relying on the calculated properties.

What does the GRAVY (hydropathy) score actually tell me about a peptide?

The GRAVY score averages the Kyte-Doolittle hydropathy value of every residue in the sequence, giving a single number that summarizes the peptide's overall water-affinity. A positive GRAVY score suggests a hydrophobic peptide that may have poor aqueous solubility, partition into membranes, or be prone to aggregation, while a negative score suggests a hydrophilic peptide that should dissolve readily in aqueous buffers. It is a useful first-pass screen before synthesis or formulation work, but it does not account for charge distribution, secondary structure, or specific hydrophobic patches, so peptides with the same overall GRAVY score can still behave very differently in practice.

Why is my peptide's extinction coefficient zero or very low?

The extinction coefficient at 280 nm depends almost entirely on the number of tryptophan and tyrosine residues in the sequence, since these are the only residues with significant UV absorbance at that wavelength under the Pace et al. method used here. A peptide with no Trp or Tyr residues will have a calculated extinction coefficient of zero, which means UV absorbance at 280 nm cannot be used to quantify that particular peptide — you would need an alternative method such as a BCA or Bradford assay, or a custom-labelled tag, to measure its concentration instead.

How accurate is the calculated molecular weight compared to mass spectrometry?

The molecular weight reported here is the average isotopic mass calculated from standard residue masses plus water for the termini, adjusted for any terminal modifications you select. This average mass is normally within a fraction of a Dalton of the value seen on a mass spectrometer for an unmodified, correctly synthesized peptide, but it will not match if the actual peptide carries unexpected modifications, incomplete deprotection, oxidation, or synthesis byproducts. For quality control of a synthesized peptide, treat this calculated value as the expected target mass to compare against your MS result, not as a substitute for actually running the spectrometry.

Can I use this calculator for peptides containing non-standard or D-amino acids?

No — this calculator only recognizes the 20 standard L-amino acids using their single-letter codes; any other character, including codes for D-amino acids, non-natural residues, or chemical linkers, will be flagged as an invalid character and excluded from the calculation. If your peptide contains such residues, the reported molecular weight, pI, and charge will all be underestimates of the true values for the modified residues' contribution. For peptides with non-standard building blocks, you will need to manually add the mass and charge contribution of each modified residue to the values calculated here.

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