How molecular weight is built from a sequence
A peptide bond forms by condensation: two amino acids join and one water molecule leaves. So a chain of n residues weighs the sum of the n free amino acids minus (n minus 1) waters, which is more conveniently expressed as the sum of the n residue masses plus one water for the free ends of the chain.
That single water, 18.02 daltons, accounts for the terminal amine hydrogen and the terminal hydroxyl. Every sequence-based weight in this toolkit is calculated that way, which is why they agree with each other.
Average mass and monoisotopic mass
Average mass uses the isotope-weighted average atomic mass of each element, which is what a balance measures and what a catalogue quotes. Monoisotopic mass uses the mass of the most abundant isotope of each element, which is what a high-resolution mass spectrometer reports for the lowest peak of the isotope cluster.
The two diverge as the molecule grows, because a larger molecule has more carbon atoms and therefore a higher chance of containing a carbon-13. For a 1,400 dalton peptide the gap is around 0.8 daltons; for a 5,000 dalton one it is closer to three. This calculator reports average mass, which is the one to compare against a specification sheet.
Why the catalogue weight often disagrees, and by how much
A weight computed from a bare sequence describes a linear peptide with a free N-terminal amine and a free C-terminal acid. Most research peptides are modified, and each modification has a fixed, recognisable mass.
When a calculated weight and a catalogue weight differ, the difference is nearly always one of a short list of numbers rather than an error in either figure.
- •C-terminal amide instead of a free acid: 0.98 Da lighter
- •N-terminal acetyl group: 42.01 Da heavier
- •Each disulfide bridge: 2.02 Da lighter
- •Pyroglutamate from an N-terminal Gln: 17.03 Da lighter
- •A single methylation: 14.02 Da heavier
- •Phosphorylation: 79.97 Da heavier
A worked example
Thymosin beta-4, sold as TB-500, has a 43 residue sequence that sums to 4,921 daltons. The catalogue figure is 4,963. The difference of 42 is one acetyl group, because the natural peptide is acetylated at the N-terminus. Neither number is wrong; they describe different molecules, one of which is the real one.
Oxytocin works the same way in the other direction. The linear sequence CYIQNCPLG comes to 1,010.19, and the published weight is 1,007.19. Subtract 2.02 for the disulfide bridge and 0.98 for the C-terminal amide and the two agree.
What single-letter code cannot express
A large fraction of synthetic research peptides contain residues that have no single-letter code at all: D-amino acids, alpha-aminoisobutyric acid, 2-naphthylalanine, unnatural or protected residues. Ipamorelin, GHRP-2 and melanotan II are all in this category.
For those, a sequence-based weight is not approximate, it is a weight for a different molecule. The honest move is to take the weight from the certificate of analysis and use the modification mass calculator to reason about deliberate changes to it.
How the molecular weight is calculated
A sum over a residue mass table plus one water. The table holds average residue masses derived from the IUPAC standard atomic weights, which is the same table used by every other sequence tool on this site.
MW = SUM( residue mass for each amino acid ) + 18.0153 worked: GHK = 57.0519 + 137.1411 + 128.1741 + 18.0153 = 340.38 Da
- Parse the sequence. Whitespace, digits and punctuation are stripped, and letters are upper-cased. Characters that are not one of the twenty standard codes are discarded and listed back to you rather than being silently ignored.
- Look up each residue. Every residue contributes its average mass in the chain form, which is the free amino acid less one water. Leucine and isoleucine are isomers and weigh the same.
- Add one water for the termini. 18.0153 daltons, covering the hydrogen on the N-terminal amine and the hydroxyl on the C-terminal acid. It is added once for the whole chain, not once per bond.
- Report the composition alongside. The per-residue counts are shown so the sum can be checked by hand, and so an unexpected weight can be traced to an unexpected residue count.
What this method cannot tell you
- •It computes the linear, unmodified, free-acid form. Terminal amides, acetyl groups, disulfide bridges and post-translational modifications all shift the real weight.
- •It cannot represent D-amino acids or non-standard residues, which have no single-letter code. A peptide containing them will return a weight for a different molecule.
- •It gives average mass, not monoisotopic mass. Compare it against a catalogue figure, not against a high-resolution mass spectrum.
- •It says nothing about salt form. A peptide supplied as a TFA or acetate salt weighs more than the free base this calculates.
Where the numbers come from
Peptide molecular weight calculator: frequently asked questions
Add the residue mass of every amino acid in the sequence, then add 18.02 daltons for the water that completes the free N-terminus and C-terminus.
For GHK: glycine 57.05, histidine 137.14, lysine 128.17, plus 18.02, giving 340.38 daltons.
Because forming a peptide bond releases a water molecule. A chain of n residues has n minus 1 bonds and has therefore lost n minus 1 waters from the n free amino acids.
Working from residue masses, which already have that water removed, means one water has to be added back for the two ends of the chain that were never joined to anything.
Almost always because the real peptide carries a modification the bare sequence does not describe. The difference is usually one of a small set of recognisable numbers.
- •0.98 Da lighter: C-terminal amide
- •42.01 Da heavier: N-terminal acetyl group
- •2.02 Da lighter per disulfide bridge
- •17.03 Da lighter: pyroglutamate from an N-terminal glutamine
If the gap matches one of these, both numbers are right and they describe different molecules.
Average mass uses the natural isotope-weighted average for each element and is what a catalogue quotes. Monoisotopic mass uses the most abundant isotope of each element and is what a high-resolution mass spectrometer reports.
They diverge as molecules get larger. Around 1,400 daltons the gap is roughly 0.8 daltons; by 5,000 daltons it is around three. This tool gives average mass.
Monoisotopic, if the instrument resolves the isotope pattern. Average, if it does not and reports a centroid of the whole envelope.
The mass spec match calculator works in monoisotopic terms and converts a neutral mass to the m/z you would actually observe at a given charge state.
Yes, exactly. They are structural isomers with the identical formula, so no mass measurement can distinguish them.
Telling them apart requires fragmentation chemistry that breaks the side chain, or independent sequence information.
Each bridge removes two hydrogen atoms, 2.02 daltons, as two cysteine thiols oxidise to a cystine linkage.
A peptide with two bridges is 4.03 daltons lighter than its fully reduced form. The disulfide bond calculator handles sequences with several cysteines and enumerates the possible pairings.
The mass is the same, because a D-amino acid is a mirror image of its L form and mirror images weigh the same.
The problem is notation, not mass: single-letter code cannot distinguish them, so a sequence containing D residues cannot be written unambiguously and the tool has no way to know they are there.
They have no single-letter code and cannot be entered. Any character that is not one of the twenty standard codes is discarded and reported back rather than silently ignored.
Peptides such as Ipamorelin, GHRP-2 and melanotan II all contain these residues, and their weights have to come from the certificate of analysis rather than from a sequence.
The peptide's own weight is unchanged, but the weight of the powder you handle is not. A TFA salt adds 114.02 daltons per counterion, and an acetate salt 60.05.
The salt form converter works out the salt weight and the resulting peptide content from the free base weight this tool gives.
A unit of mass equal to one twelfth of the mass of a carbon-12 atom, roughly the mass of one hydrogen atom. It is numerically the same as grams per mole.
A peptide of 1,000 daltons weighs 1,000 grams per mole, which means one millimole of it is one gram.
The boundary is conventional, not physical. Around 50 residues is the usual dividing line, which is roughly 5,500 daltons.
Nothing changes chemically at that point. The sequence length calculator shows the classification scale and where a given sequence falls on it.
Because only the twenty standard single-letter codes correspond to a residue mass. Spaces, numbers, hyphens and the ambiguity codes B, J, O, U, X and Z are all removed.
Anything discarded is listed under the input, so a typo shows up rather than quietly changing the answer.
Yes. The header line and any line breaks are stripped along with the other non-residue characters, so a pasted FASTA block parses correctly.
To within a few hundredths of a dalton for the molecule as described. The residue table carries four decimal places, and the rounding difference against a catalogue figure is normally in the last digit.
A discrepancy larger than a dalton is a modification or a different molecule, not a rounding error.
Its sequence GEPPPGKPADDAGLV gives 1,419.5 daltons as a linear free acid, which matches the commonly quoted 1,419.53.
Unlike many research peptides, BPC-157 is unmodified at both termini, which is why the sequence calculation and the catalogue figure agree directly.
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