Peptide properties, from the sequence.

Residues, groups, scales

Mass

M = Σ M(residue) + M(H₂O) [+ C₂H₂O acetyl] [− O + N + H amide] [− 2 H per disulfide]

Residue = amino acid less H₂O. Average masses: IUPAC 2021 standard atomic weights (C 12.011, H 1.008, N 14.007, O 15.999, S 32.06). Monoisotopic: ¹²C 12, ¹H 1.007 825, ¹⁴N 14.003 074, ¹⁶O 15.994 915, ³²S 31.972 071.

Charge

z(pH) = Σ_basic 1 / (1 + 10^(pH − pK)) − Σ_acidic 1 / (1 + 10^(pK − pH))

Each group independently (Henderson–Hasselbalch). Bjellqvist pK: D 4.05 · E 4.45 · H 5.98 · C 9.00 · Y 10.00 · K 10.00 · R 12.00; N-terminus 7.50 (A 7.59, M 7.00, S 6.93, P 8.36, T 6.82, V 7.44, E 7.70); C-terminus 3.55 (D 4.55, E 4.75).

pI

z(pI) = 0

Found by bisection between pH 0 and 14. Undefined when the charge never crosses zero, a peptide with no acidic or no basic group.

GRAVY

GRAVY = Σ hydropathy / N

The mean Kyte–Doolittle hydropathy of the residues: I 4.5 … R −4.5. Positive reads hydrophobic.

Aromaticity

(nF + nW + nY) / N

Lobry and Gautier 1994: the fraction of aromatic residues.

ε₂₈₀

ε = 5 500 nW + 1 490 nY + 125 n(cystine) M⁻¹·cm⁻¹ ; A(0.1 %) = ε / M

Pace et al. 1995. The reduced value leaves the cystine term out; A(0.1 %) is the absorbance of 1 mg/mL in a 1 cm cell.

Against the literature

Average masses computed here against the molecular weights suppliers and databases quote; the small differences are the atomic-weight tables used.

What the numbers mean

Average or monoisotopic mass?

The average mass uses the natural isotope mixture of each element, the IUPAC standard atomic weights, and is what a balance, a supplier's certificate and most databases quote. The monoisotopic mass is that of the species made only of the lightest isotopes and is what the first peak of a high-resolution mass spectrum shows. For a 1 to 3 kDa peptide they differ by roughly 0.6 to 1.3 Da.

Which pK values, and why does the pI differ from other tools?

The Bjellqvist set, the one ExPASy Compute pI/Mw uses, with its residue-specific terminal values. Other tools use the Lehninger, EMBOSS or IPC sets, and differences of a few tenths of a pH unit between them are normal. Every set treats the groups as independent, so the result is an estimate; for short peptides, where the termini dominate, the estimate is roughest.

How are the modifications handled?

An N-terminal acetyl removes the terminal amine from the charge model and adds C₂H₂O to the mass. A C-terminal amide replaces the terminal OH by NH₂ (−0.98 Da) and removes the carboxylate. Each disulfide removes two hydrogens and takes two cysteines out of the ionizable set. Nothing else is modeled: a D-residue has the same mass and charge as its L form, so it passes through unchanged, but chelators such as DOTA, labels, PEG and cyclization are outside the tool.

What are GRAVY, aromaticity and ε₂₈₀ for?

GRAVY, the mean Kyte–Doolittle hydropathy, is a first guide to how hydrophobic a peptide is: positive values suggest poor water solubility and membrane affinity. Aromaticity is the share of Phe, Trp and Tyr. The extinction coefficient at 280 nm lets a peptide be quantified by absorbance, but only Trp, Tyr and cystine contribute, so a peptide without them cannot be measured that way.

• Prohaska T et al. Standard atomic weights of the elements 2021 (IUPAC Technical Report). Pure Appl Chem 2022;94:573–600 (opens in a new tab) — the average atomic weights
• NIST — Atomic Weights and Isotopic Compositions (relative atomic masses) (opens in a new tab) — the monoisotopic masses
• Bjellqvist B et al. The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 1993;14:1023–1031 (opens in a new tab) — the pK set
• Bjellqvist B et al. Reference points for comparisons of two-dimensional maps of proteins from different human cell types. Electrophoresis 1994;15:529–539 (opens in a new tab) — the terminal pK refinements
• Gasteiger E et al. Protein identification and analysis tools on the ExPASy server. The Proteomics Protocols Handbook, 2005 (opens in a new tab) — Compute pI/Mw method
• Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982;157:105–132 (opens in a new tab) — the hydropathy scale, GRAVY
• Pace CN et al. How to measure and predict the molar absorption coefficient of a protein. Protein Sci 1995;4:2411–2423 (opens in a new tab) — ε at 280 nm
• Lobry JR, Gautier C. Hydrophobicity, expressivity and aromaticity are the major trends of amino-acid usage in 999 Escherichia coli chromosome-encoded genes. Nucleic Acids Res 1994;22:3174–3180 (opens in a new tab) — aromaticity

The other six, open in the browser.

• Kd to pKd and ΔGCalculator→
• Decay and activity plannerCalculator→
• Molar and specific activityCalculator→
• SUV calculatorCalculator→
• Structure viewer3D viewer→
• Isotope half-livesReference table→