For reference: +4 · 4731.3 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.
Can someone walk through the arithmetic step by step?
m/z = 1183.83 at 4+. Electrospray charges a peptide by adding protons, so the observed ion is the neutral mass plus 4 protons, all divided by the charge: (4731.3 + 4 × 1.00728) ÷ 4 = 4735.329 ÷ 4 = 1183.83. The proton term is the one people drop, and because it is z protons over z charges it shifts m/z by 1.007 at every charge state — small, and far larger than the mass accuracy of the instrument. The neighbouring charge state sits at 947.27, and seeing the two of them where they belong is better identity evidence than either one alone. Use the average mass against an average-mass calculation and the monoisotopic mass against a monoisotopic one; mixing them costs you a couple of daltons on a peptide this size.
The underlying point is that 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.
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.
| Test | Answers | Does NOT answer |
|---|---|---|
| RP-HPLC, area % | What fraction of detected material is the target | How much target is present |
| Quantified content | Milligrams of peptide per vial | What the impurities are |
| ESI-MS identity | Whether the molecular weight matches | Purity, or isomeric substitution |
| Peptide mapping | Sequence, localised to a fragment | Quantity |
| Karl Fischer | Water content of the solid | Solvent content |
| LAL endotoxin | Pyrogen load in EU/mg | Sterility |
| Sterility test | Growth in defined media over 14 days | Endotoxin, or bioburden count |
A mass shift of minus eighteen usually means dehydration or a succinimide intermediate, which is pH-dependent and can be reversible.
Electrospray ionisation soft-ionisation behaviour is well-characterised and standards exist for m/z calibration and mass accuracy assessment.
Always run a blank between samples and check for carry-over.
Analytical standards and reagents with traceable certificates. Every quantitative result you read inherits the accuracy of the standard behind it.
Shop standardsAsk PeptideStack is a static archive. Posting is closed, but the norms are worth stating: answer the question that was asked, show your working, cite the trial or the certificate, and say plainly where the evidence runs out.