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Peptide Calculator: pI, Charge, Mass and ε280
In short: Enter a peptide sequence. You get the average and monoisotopic mass, the formula, the isoelectric point with four pKa sets, the net charge at any pH with a curve, ε280, GRAVY, the instability and aliphatic index and a hydropathy plot. It is free and runs in your browser.
How to use the peptide calculator
- Type the sequence in one-letter code (
DRVYIHPF) or three-letter code (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). Write phosphorylated residues aspS,pT,pYorpSer,pThr,pTyr. - Tick N-terminal acetyl or C-terminal amide, or write
Ac-and-NH2in the sequence. Tick disulfides if the cysteines are paired. - Choose a pKa set and the pH for the net charge. Bjellqvist gives the same pI as ExPASy ProtParam.
- Read the results, hover over the curves, then copy the summary or download a CSV with the charge-vs-pH table.
Worked examples (Bjellqvist pKa set)
| Peptide | Average / mono mass (Da) | pI | ε280 (M⁻¹cm⁻¹) | GRAVY | Instab. | Aliph. |
|---|---|---|---|---|---|---|
| Angiotensin II, DRVYIHPF | 1046.19 / 1045.5345 | 6.74 | 1490 | −0.325 | 25.80 | 85.00 |
| Insulin B chain, FVNQHLCGSHLVEALYLVCGERGFFYTPKT | 3429.96 / 3427.6845 | 6.90 | 2980 (3105 as cystine) | 0.220 | 9.85 | 84.33 |
| Glucagon, HSQGTFTSDYSKYLDSRRAQDFVQWLMNT | 3482.79 / 3480.6157 | 6.75 | 8480 | −0.990 | 56.03 | 40.34 |
| Human ubiquitin (76 aa) | 8564.84 / 8559.6166 | 6.56 | 1490 | −0.489 | 36.06 | 100.00 |
Instab. = instability index; Aliph. = aliphatic index. Values were cross-checked against Biopython's ProtParam module, which implements the ExPASy definitions. The masses match the protein molecular weight calculator.
How the values are calculated
Mass and formula
Mass is the sum of the residue masses plus one water (18.01524 Da average, 18.010565 Da monoisotopic). Acetyl adds C2H2O (+42.0106 Da mono), amide swaps OH for NH2 (−0.9840 Da), phosphate adds HPO3 (+79.9663 Da) and each disulfide removes 2 H (−2.0157 Da).
Net charge and pI
z(pH) = Σbasic 1 / (1 + 10pH − pKa) − Σacidic 1 / (1 + 10pKa − pH)
The pI is the pH where z = 0, found by bisection between pH 0 and 14. Disulfide-bonded Cys, an acetylated N-terminus and an amidated C-terminus carry no charge. Each phosphate adds two acidic groups (pKa about 2.1 and 5.8–5.9, the same in every set).
| pKa set | N-term | C-term | Asp | Glu | His | Cys | Tyr | Lys | Arg |
|---|---|---|---|---|---|---|---|---|---|
| Bjellqvist (ExPASy)* | 7.5 | 3.55 | 4.05 | 4.45 | 5.98 | 9.0 | 10.0 | 10.0 | 12.0 |
| EMBOSS | 8.6 | 3.6 | 3.9 | 4.1 | 6.5 | 8.5 | 10.1 | 10.8 | 12.5 |
| Lehninger | 9.69 | 2.34 | 3.86 | 4.25 | 6.0 | 8.33 | 10.0 | 10.5 | 12.4 |
| Thurlkill 2006 | 8.0 | 3.67 | 3.67 | 4.25 | 6.54 | 8.55 | 9.84 | 10.4 | 12.3 |
* Bjellqvist also uses terminal values that depend on the end residue: N-terminal Ala 7.59, Met 7.0, Ser 6.93, Pro 8.36, Thr 6.82, Val 7.44, Glu 7.7; C-terminal Asp 4.55, Glu 4.75. The default is Bjellqvist because it reproduces ExPASy, the value most people compare with. The EMBOSS set is the one used by the sequence to structure tool; Thurlkill 2006 (alanine pentapeptides) is the set used by the protonation state tool. Lehninger values are for free amino acids, so they overstate the N-terminal pKa of a peptide.
Extinction coefficient at 280 nm
ε280 = 5500 n(Trp) + 1490 n(Tyr) + 125 n(cystine) A280(1 mg/mL) = ε / MW
Values from Pace et al., Protein Science 1995, 4, 2411. Two values are given: all Cys paired as cystines, and all Cys reduced.
GRAVY, instability index and aliphatic index
- GRAVY: mean Kyte–Doolittle hydropathy of all residues (Kyte & Doolittle, J. Mol. Biol. 1982, 157, 105).
- Instability index: (10 / L) × Σ DIWV(xi, xi+1) over all dipeptides (Guruprasad, Reddy & Pandit, Protein Eng. 1990, 4, 155). Under 40 is classed as stable.
- Aliphatic index: mol% Ala + 2.9 × mol% Val + 3.9 × (mol% Ile + mol% Leu) (Ikai, J. Biochem. 1980, 88, 1895).
- Hydropathy plot: Kyte–Doolittle mean over a sliding window, plotted at the window centre. With a 19-residue window, stretches above 1.6 are possible transmembrane segments.
Limitations
- The pI and charge use fixed pKa values for each group. Real values shift with neighbouring charges, folding, salt and temperature, so a measured pI can differ by 0.5 units or more. Different sets disagree for the same reason.
- Only the 20 standard amino acids, N-acetyl, C-amide, pSer, pThr, pTyr and disulfides are handled. D-amino acids give the same numbers as L. Other modifications (lipids, glycans, cyclisation, non-natural residues) are not supported.
- ε280 from residue counts is accurate to about 5% for folded proteins and less for short peptides. Measure it if you need exact concentrations.
- The instability index, GRAVY and aliphatic index are statistical indicators derived from proteins, not measured properties of your peptide.
- Monoisotopic mass is for the neutral molecule; [M+H]⁺ and [M+2H]²⁺ add 1.007276 Da per proton.
Frequently asked questions
How is the isoelectric point of a peptide calculated?
Each ionisable group (N-terminus, C-terminus, Asp, Glu, Cys, Tyr, His, Lys, Arg) carries a fractional charge from the Henderson–Hasselbalch equation. The sum is the net charge. The pI is the pH where the sum is zero, found here by bisection between pH 0 and 14.
Why do different pI calculators give different values?
They use different pKa values. For ubiquitin the Bjellqvist set used by ExPASy gives pI 6.56, while the EMBOSS set gives 7.54. No set is exact, because real pKa values depend on the neighbours and the fold. The tool shows all four sets side by side.
Does this match ExPASy ProtParam?
With the Bjellqvist pKa set the pI, average mass, extinction coefficients, GRAVY, instability index and aliphatic index follow the ProtParam definitions. Ubiquitin gives 8564.84 Da, pI 6.56, GRAVY −0.489 and aliphatic index 100.00.
How do N-terminal acetylation and C-terminal amidation change the result?
Acetylation adds C2H2O (+42.011 Da monoisotopic) and removes the positive N-terminal amine. Amidation replaces OH by NH2 (−0.984 Da) and removes the negative C-terminal carboxylate. Both change the pI, often by more than one unit for short peptides.
What do the instability index and GRAVY mean?
The instability index (Guruprasad 1990) sums weights of dipeptides; a value under 40 predicts a stable protein in a test tube. GRAVY is the average Kyte–Doolittle hydropathy; positive values mean a hydrophobic sequence. Both are statistical rules of thumb, not measurements.
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