Setup, so nobody has to ask: +3 · 4813.5 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?
m/z = 1605.51 at 3+. Electrospray charges a peptide by adding protons, so the observed ion is the neutral mass plus 3 protons, all divided by the charge: (4813.5 + 3 × 1.00728) ÷ 3 = 4816.522 ÷ 3 = 1605.51. 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 1204.38, 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.
Two 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.
Mechanically, 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.
Always run a blank between samples and check for carry-over.
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Browse resultsIt helps to be literal here: 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 eighteen usually means dehydration or a succinimide intermediate, which is pH-dependent and can be reversible.
The part that matters: 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.
Electrospray ionisation soft-ionisation behaviour is well-characterised and standards exist for m/z calibration and mass accuracy assessment.
The practical summary: use mass spectrometry for identity, not for purity.
More usefully, 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.
The m/z accuracy achievable depends on the mass analyser type — quadrupole gives low accuracy, time-of-flight gives moderate accuracy, and Orbitrap gives high accuracy.
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.
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.
Specifically, tandem mass spectrometry fragments the ions and measures the fragment masses, which provides sequence information and is the best tool for confirming identity.
High-resolution mass spectrometry can distinguish a Lys-containing peptide from an Arg-containing peptide of similar mass because of the isotope difference.
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.
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.
edited 20 Aug 2024 by laminar_bench — added the placebo-arm figures
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.
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.
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.