Type or paste a peptide sequence in one-letter (YGGFL) or three-letter code (Tyr-Gly-Gly-Phe-Leu), or build it with the amino acid buttons. The calculator gives the formula, average and monoisotopic mass, net charge, isoelectric point and extinction coefficient, and converts the sequence between the two codes. Spaces, numbers and line breaks are ignored.
Mass and formula
The formula is the sum of the residues plus one molecule of water for the free ends: each peptide bond releases one water, so a chain of n residues has lost n − 1 of them. A C-terminal amide replaces –OH with –NH2 and lowers the mass by 0.98 Da. An N-terminal acetyl group adds C2H2O, 42.04 Da. Each disulfide bond removes two hydrogen atoms, 2.02 Da.
The calculator gives two masses because they answer different questions. The average mass uses the conventional atomic weights recommended by IUPAC (H 1.008, C 12.011, N 14.007, O 15.999, S 32.06)[1], which reflect the natural mix of isotopes. Use it to weigh a peptide and make up solutions. The monoisotopic mass counts only the lightest common isotopes, 12C, 1H, 14N, 16O and 32S[2]. A mass spectrometer measures the mass-to-charge ratio (m/z) of ions and separates those that differ by one isotope. Once the charge and any attached protons or other ions are taken into account, the monoisotopic mass corresponds to the first peak of a small peptide's isotope pattern.
A freeze-dried peptide is almost never pure peptide: it also holds counter-ions, often trifluoroacetate from purification, and water. The weighed amount is therefore larger than the amount of peptide.
Charge and isoelectric point
Each ionisable group carries a fraction of a charge that depends on the pH and on its pKa. The groups counted are the free N-terminal amine, the free C-terminal carboxyl and the side chains of Lys, Arg and His (positive) and Asp, Glu, Cys and Tyr (negative). The calculator uses the pKa values of the EMBOSS package[3]: 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. Cysteines joined in disulfide bonds are not counted, and neither are capped ends.
The isoelectric point is the pH at which the net charge is zero. Here it is found numerically to 0.01 pH units. In a real peptide each pKa is shifted by its neighbours, and other programs use other sets of values, so different calculators give somewhat different pI values for the same sequence. The figure is a guide for choosing buffers and separation conditions, not a measurement.
Extinction coefficient
The molar extinction coefficient at 280 nm lets you measure a peptide's concentration from its absorbance. It is estimated from the number of tryptophans, tyrosines and disulfide bonds (cystines) with the equation of Pace and co-workers, fitted to 116 measured values for 80 folded proteins in water[4]:
ε280 = 5500 × nTrp + 1490 × nTyr + 125 × ncystine M−1 cm−1
Concentration is then A280 / (ε × path length). Free cysteines do not absorb significantly, so they add nothing. The estimate is quite reliable when the sequence contains tryptophan and less so when it does not; the authors advise measuring the coefficient when accuracy matters[4]. A peptide with no tryptophan or tyrosine absorbs very little at this wavelength, even if it has disulfide bonds, which add only 125 each, and its concentration has to be measured another way.
References
- Meija J, Coplen TB, Berglund M, et al. (2016). Atomic weights of the elements 2013 (IUPAC Technical Report). Pure and Applied Chemistry 88:265–291. DOI: 10.1515/pac-2015-0305
- Wang M, Audi G, Kondev FG, Huang WJ, Naimi S, Xu X (2017). The AME2016 atomic mass evaluation (II). Tables, graphs and references. Chinese Physics C 41:030003. DOI: 10.1088/1674-1137/41/3/030003
- Rice P, Longden I, Bleasby A (2000). EMBOSS: the European Molecular Biology Open Software Suite. Trends in Genetics 16:276–277. DOI: 10.1016/S0168-9525(00)02024-2. The pKa values are from the data file of the EMBOSS program iep (documentation).
- Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995). How to measure and predict the molar absorption coefficient of a protein. Protein Science 4:2411–2423. DOI: 10.1002/pro.5560041120