The specifics, since they change the answer: +4 · 3751.0 Da.
I would rather understand the derivation than memorise the outcome.
Two people I asked gave two answers that differ by a factor of ten, which is suggestive.
What is the general form of this calculation?
Concretely, tandem mass spectrometry fragments the ions and measures the fragment masses, which provides sequence information and is the best tool for confirming identity.
Electrospray ionisation creates multiple charge states of the same peptide — a 4 kDa peptide might appear at +2, +3 and +4 — and all of them must be accounted for in the spectrum.
| 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 |
Stated carefully, deconvolution of a mass spectrum with multiple charge states produces a reconstructed neutral mass, and errors in the deconvolution produce errors in the inferred mass.
False positives from contamination are common in mass spectrometry work, and running a blank between every sample and a solvent background are standard practice.
The practical summary: use mass spectrometry for identity, not for purity.
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Browse resultsMore usefully, start from what electrospray ionisation does: it ionises the peptide without fragmenting it, creating singly or multiply charged species that the mass analyser then separates by their mass-to-charge ratio.
A mass shift of minus eighteen usually means dehydration or a succinimide intermediate, which is pH-dependent and can be reversible.
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.
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.
It helps to be literal here: identity confirmation from mass spectrometry means matching the observed m/z to the calculated m/z for your peptide at its known charge states.
High-resolution mass spectrometry can distinguish a Lys-containing peptide from an Arg-containing peptide of similar mass because of the isotope difference.
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.
A correct mass is necessary for identity but not sufficient — you also need the chromatography to confirm it.
More usefully, coupling HPLC to a mass spectrometer adds identity information to the chromatographic separation, but the mass spectrometer's ionisation conditions can distort the HPLC peak shape.
A mass shift of minus one hundred and twenty-eight usually means a missing Gln or Lys residue from a synthesis deletion sequence.
Peptide mapping — enzymatic digestion followed by tandem mass spectrometry — can confirm the primary sequence and is the method of choice when identity is ambiguous.
Worth noting that source contamination is common and silent, so a result that looks too good to be true often is.
The practical summary: use mass spectrometry for identity, not for purity.
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.
A mass shift of plus one usually means deamidation at asparagine or glutamine, which creates a secondary amine instead of an amide and changes the mass by exactly one.
Always run a blank between samples and check for carry-over.
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