Setup, so nobody has to ask: +3 · 4207.2 Da.
The units are where I keep going wrong, so please be explicit about them.
I have sanity-checked the order of magnitude and it seems right, which is not the same as being right.
Where is my error, and what is the correct working?
On the detail: a mass shift of exactly zero with a shifted retention time points to an isomer — a scrambled disulfide or a racemised residue — which mass spectrometry alone cannot identify.
For a large peptide with multiple peaks in the mass spectrum, comparing the observed isotope pattern to the calculated pattern is a quick check that the formula matches.
| Component | Typical share | Counted in purity? | Counted in content? |
|---|---|---|---|
| Target peptide | 88–94 % | Yes, as main peak | Yes |
| Related impurities | 1–3 % | Yes, as other peaks | No |
| Counter-ion (TFA or acetate) | 2–8 % | No | No |
| Residual water | 2–6 % | No | No |
| Bulking agent, if present | 0–40 % | No | No |
It helps to be literal here: a monoisotopic mass includes only the lightest isotope of each element, while the average mass weights by natural isotope abundance, and small peptides use monoisotopic mass.
I would not trust a mass result without a good baseline and a blank injection check.
A correct mass is necessary for identity but not sufficient — you also need the chromatography to confirm it.
edited 20 Mar 2026 by plate_count_9k — updated for the 2026 guidance change
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Browse resultsWorth being precise here: scrambled disulfides have the same mass as correctly formed ones, so mass spectrometry alone cannot detect a scrambling failure.
A mass shift of minus eighteen usually means dehydration or a succinimide intermediate, which is pH-dependent and can be reversible.
More usefully, high-resolution mass spectrometry can distinguish a Lys-containing peptide from an Arg-containing peptide of similar mass because of the isotope difference.
The caveat is that a correct mass does not mean the peak is correct — isomers and co-eluting species can have the same m/z.
Always run a blank between samples and check for carry-over.
Stated carefully, a D-amino-acid substitution has the same molecular weight as the L-form, so mass spectrometry cannot distinguish them without fragmenting the peptide.
A mass shift of minus one hundred and twenty-eight usually means a missing Gln or Lys residue from a synthesis deletion sequence.
Mechanically, the baseline noise on a mass spectrum sets the limit of detection, and a weak signal close to the noise is not reliable evidence for the presence of a species.
Peptide mapping — enzymatic digestion followed by tandem mass spectrometry — can confirm the primary sequence and is the method of choice when identity is ambiguous.
The practical summary: use mass spectrometry for identity, not for purity.
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