Peptide analyser
Mass, isoelectric point, extinction coefficient and composition from a sequence.
FASTA headers, whitespace, digits and lower case are all fine. Ambiguity codes are not — they have no single mass or pKa.
Match this one when you need to agree with ExPASy Compute pI/Mw, which most published pI values are quoted from.
The 280 nm coefficient counts disulfide bonds, not cysteines.
Length
Average mass
Isoelectric point
C409H619N105O108S2 · monoisotopic 8793.5614 · GRAVY -0.004 · aliphatic index 88.4
Net charge against pH
+0.358net charge
Absorbance at 280 nm
ε = 8480 M⁻¹cm⁻¹ · A(1 mg/mL, 1 cm) = 0.9637
Enter a reading
Composition · 20 of 20 present
- A Ala79.33%
- R Arg45.33%
- N Asn22.67%
- D Asp34%
- C Cys11.33%
- Q Gln34%
- E Glu68%
- G Gly22.67%
- H His34%
- I Ile22.67%
- L Leu810.7%
- K Lys68%
- M Met11.33%
- F Phe912%
- P Pro11.33%
- S Ser56.67%
- T Thr22.67%
- W Trp11.33%
- Y Tyr22.67%
- V Val79.33%
M = Σ residues + H₂O; ε₂₈₀ = 5500·nW + 1490·nY + 125·nSS; pI where Σq(pH) = 0- Extinction coefficient at 280 nm from sequence — Analytical Biochemistry, 1989
- Bjellqvist pKa set, as used by ExPASy Compute pI/Mw — Electrophoresis, 1993
- Hydropathy scale used for GRAVY — Journal of Molecular Biology, 1982
Masses use IUPAC atomic weights and may differ from a supplier’s last digit. The extinction coefficient is for the denatured protein in water; a folded protein in buffer typically reads a few per cent lower.
When to use this
Use this to get mass, isoelectric point, extinction coefficient and composition from a protein sequence. The extinction coefficient is what turns an A280 reading into a concentration; the pI is the least reliable output and should be treated as a starting point rather than a measurement.
Worked example
You have a purified protein and an A280 reading of 0.55.
- Sequence
- Your protein, 1 Trp and 2 Tyr
- Cysteines
- Reduced
- pKa set
- Bjellqvist (ExPASy)
Result
A280 of 0.55 through 1 cm is therefore about 65 µM.
What people get wrong
- Guessing the cysteine state. The 280 nm coefficient counts disulfide bonds, not cysteines, so reduced and oxidised protein give different answers and different concentrations.
- Quoting a computed pI as though it were measured. It depends on the pKa set, and it assumes every ionisable group is freely exposed, which in a folded protein they are not.
- Forgetting the tag. A His tag, a cleaved signal peptide or a phosphorylation all change the real molecule, and phosphorylation moves the pI substantially.
Questions
+Why do two tools give different pI values?
Different pKa sets. Bjellqvist matches ExPASy, which is where most published values come from; EMBOSS is used by pipelines built on it. The tool lets you pick.
+My protein has no tryptophan — can I still use A280?
Cautiously. With tyrosine alone the coefficient is least reliable, and with neither residue the protein does not absorb at 280 at all. Use a colourimetric assay instead.
+Average or monoisotopic mass?
Average for ordinary use. Monoisotopic for mass spectrometry, where the difference is a whole dalton on a small protein. Both are reported.
Related tools
- Molecular weight calculator — Compute molar mass from a chemical formula, including hydrates.
- DNA translation — Translate a nucleotide sequence into protein in any reading frame.
- Molarity calculator — Convert between mass, molar concentration and volume for a solution.
Science last reviewed .