Protein Molecular Weight Calculator

How to Use the Protein Molecular Weight Calculator

1. 1. Paste the sequence. One-letter codes, plain or FASTA. Spaces, numbers, line breaks and lower case are ignored; the 20 standard residues plus selenocysteine (U) and pyrrolysine (O) are recognised, while ambiguous codes B, J, X and Z are left out and reported.
2. 2. Set the chemistry. Tick N-terminal acetylation or C-terminal amidation if the peptide has them, and enter the number of disulfide bonds. Each bond removes two hydrogens from the mass, lets you count cystines in the extinction coefficient, and takes two cysteines out of the charge.
3. 3. Choose the pKa set and the pH. The pI and the net charge use the Henderson–Hasselbalch equation with the Bjellqvist or EMBOSS values.
4. 4. Read the results. Average mass, monoisotopic mass and [M+H]⁺, the formula, the two extinction coefficients with the absorbance of a 1 mg/mL solution, and the composition. With several FASTA records you also get a summary table.

Average and Monoisotopic Mass

A protein's mass is the sum of its residue masses plus one water for the free termini. Each residue is an amino acid minus water, so its formula is fixed — glycine is C₂H₃NO, tryptophan C₁₁H₁₀N₂O. The average mass weights every element by its natural isotope mix and is what you want for a gel, a size-exclusion column or a molar concentration. The monoisotopic mass uses only the lightest, most abundant isotope of each element (¹²C, ¹H, ¹⁴N, ¹⁶O, ³²S) and is what a high-resolution mass spectrometer resolves for peptides; the tool also gives the singly protonated ion, [M+H]⁺ = M + 1.00728. For large proteins the isotope peaks merge and the average mass is the useful one.

Average masses use C 12.0107, H 1.00794, N 14.0067, O 15.9994, S 32.065; other tools use slightly different atomic weights, so the second decimal can differ. Results agree with Biopython's molecular_weight and ProteinAnalysis for the test proteins. Isoleucine and leucine have identical masses, which is why mass spectrometry cannot tell them apart.

Extinction Coefficient at 280 nm

Tryptophan, tyrosine and, weakly, cystine absorb at 280 nm. Pace and colleagues (1995, Protein Science 4:2411–2423) give ε₂₈₀ = 5,500 × (Trp) + 1,490 × (Tyr) + 125 × (cystine) in M⁻¹ cm⁻¹, measured in 6 M guanidinium chloride, which unfolds the protein. The tool reports it with every cysteine reduced and with cystines counted — the number of disulfide bonds you entered or, if you entered none, every pair of cysteines assumed bonded, as ExPASy does. Divide ε by the molecular weight for the absorbance of a 1 mg/mL solution; dividing a measured A₂₈₀ by ε gives the molar concentration by Beer–Lambert (A = ε·c·l). The estimate is poor for proteins with few aromatic residues and can differ from the value measured on the folded protein by 10% or more.

Isoelectric Point and Net Charge

The net charge at a pH is the sum, over the N-terminus, C-terminus and the ionisable side chains (Lys, Arg, His, Asp, Glu, Cys, Tyr), of each group's fractional charge from the Henderson–Hasselbalch equation, 1 / (1 + 10^{±(pH − pK)}). The pI is the pH where that sum is zero. The result depends on the pKa set: Bjellqvist (Bjellqvist et al., 1993 and 1994, derived from immobilised pH gradient gels and used by ExPASy and Biopython) also varies the terminal pK with the first and last residue, while EMBOSS iep uses a single value per group (N-terminus 8.6, C-terminus 3.6, Lys 10.8, Arg 12.5, His 6.5, Asp 3.9, Glu 4.1, Cys 8.5, Tyr 10.1). Predicted pI values are less reliable for very basic proteins and for short peptides.

Worked Example: Hen Egg-White Lysozyme

Try an example loads mature hen egg-white lysozyme (UniProt P00698, residues 19–147): 129 residues with 8 cysteines paired in 4 disulfide bonds, and the number of bonds is set to 4.

The first column is what sequence-only tools such as ExPASy and UniProt report. The second matches the 14.3 kDa that mass spectrometry finds for the intact enzyme. The predicted pI changes by more than a pH unit once the eight cysteines are treated as four disulfides, because cystine does not ionise, and it moves toward the value of about 11 reported for lysozyme in experiments. If your protein has disulfides, entering their number is worth doing before you quote a pI. Get the same protein's sequence from DNA with the DNA to protein translator, or compare two sequences with the sequence alignment tool.

Frequently Asked Questions

How do you calculate the molecular weight of a protein from its sequence?

Add up the mass of each amino acid residue (the amino acid minus a water) and add one water for the two ends. For a 129-residue lysozyme that gives about 14,313 Da. Use average residue masses for the molecular weight and monoisotopic masses when you need the mass a mass spectrometer would resolve.

What is the difference between average and monoisotopic mass?

Average mass weights each element by its natural isotope abundances; monoisotopic mass uses only the most abundant isotope of each. For a small peptide the monoisotopic peak is the strong, resolved one; for a large protein the molecule almost never contains only light isotopes, so the average is the meaningful figure.

How accurate is the predicted isoelectric point?

It is an estimate. The calculation ignores the protein's folding, neighbouring charges and ionic strength, and pKa values shift inside a protein. Predictions are often within about one pH unit of a measured pI, and worse for very basic proteins and short peptides. Counting disulfide-bonded cysteines correctly matters for cysteine-rich proteins.

How do I use the extinction coefficient to find protein concentration?

Measure A₂₈₀ in a 1 cm cuvette and divide by ε in M⁻¹ cm⁻¹ to get the concentration in mol/L; multiply by the molecular weight for g/L. Alternatively divide A₂₈₀ by the A₂₈₀ of 1 mg/mL given by the tool to get mg/mL. Correct for light scattering and for nucleic acid, which also absorbs near 280 nm.

Why are some amino acid codes left out?

B, J, X and Z stand for more than one residue, so they have no single mass. They are skipped and counted in a notice, which means the calculated mass applies to the remaining residues only. Replace them with a specific residue if you know it.

Does it include the mass of post-translational modifications?

Only the three adjustments offered: disulfide bonds, N-terminal acetylation and C-terminal amidation. Phosphorylation, glycosylation, signal peptide removal and other modifications must be applied to the sequence or the mass yourself.