The case in front of me: +5 · 4813.5 Da.
I want the working, not the result — I need to be able to redo it with different numbers.
I care about the precision as well as the value — I want to know how many figures are real.
How many significant figures are actually justified here?
m/z = 963.71 at 5+. Electrospray charges a peptide by adding protons, so the observed ion is the neutral mass plus 5 protons, all divided by the charge: (4813.5 + 5 × 1.00728) ÷ 5 = 4818.536 ÷ 5 = 963.71. 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 803.26, 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.
More usefully, the single most important fact about mass spectrometry for peptides is that it measures only the molecular weight and tells you almost nothing about whether the peak is actually your target.
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.
| 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 |
Mechanically, 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.
Peptide mapping — enzymatic digestion followed by tandem mass spectrometry — can confirm the primary sequence and is the method of choice when identity is ambiguous.
One qualification: high-resolution mass spectrometry gives high mass accuracy but low speed, and the reverse is true for low-resolution instruments.
A correct mass is necessary for identity but not sufficient — you also need the chromatography to confirm it.
Analytical standards and reagents with traceable certificates. Every quantitative result you read inherits the accuracy of the standard behind it.
Shop standardsTwo ions with the same nominal mass but different molecular formulae have different exact masses, and only high-resolution mass spectrometry can distinguish them.
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.
Stated carefully, 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.
Electrospray ionisation soft-ionisation behaviour is well-characterised and standards exist for m/z calibration and mass accuracy assessment.
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.
edited 30 Dec 2024 by mz_4113 — added the placebo-arm figures
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.
High-resolution mass spectrometry can distinguish a Lys-containing peptide from an Arg-containing peptide of similar mass because of the isotope difference.
A mass shift of minus eighteen usually means dehydration or a succinimide intermediate, which is pH-dependent and can be reversible.
The limitation is that mass spectrometry tells you the mass and almost nothing else, so it needs to be paired with chromatography or other identity information.
Always run a blank between samples and check for carry-over.
The underlying point is that tandem mass spectrometry fragments the ions and measures the fragment masses, which provides sequence information and is the best tool for confirming identity.
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.
False positives from contamination are common in mass spectrometry work, and running a blank between every sample and a solvent background are standard practice.
If you only pay for one test, pay for quantified content. Purity is the number everyone quotes and content is the number that changes what you do.
Stated carefully, identity confirmation from mass spectrometry means matching the observed m/z to the calculated m/z for your peptide at its known charge states.
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.
In practice: ask for the chromatogram, check the method section, check the lot number against the vial, and set your accept threshold before you see the result rather than after.
Ask 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.