Mass spectrometry on a peptide COA: confirming identity, not purity

What the MS line on a certificate is for

Most peptide certificates pair an HPLC purity percentage with a mass spectrometry result. Buyers often read the two as one quality score. They are separate tests. HPLC reports how much of the detected signal sits in the main peak. Mass spectrometry reports the mass-to-charge ratio (m/z) of ions the instrument detected, and the certificate compares that with the mass expected for the ordered sequence.

Suppliers describe the split the same way. Peptides EU, for example, says its purity is measured by reverse-phase HPLC and identity is confirmed by mass spectrometry. [6] Treat the MS line as an identity check, and read it with three questions: which mass was expected, which ion was observed, and how close the two are.

Compute the expected mass from the sequence

A peptide's monoisotopic mass is the sum of its residue masses plus one water (H2O) for the free N- and C-termini. Each residue mass comes from its elemental formula and the masses of the most abundant isotopes: 1H 1.00782503223, 12C 12 exactly, 14N 14.00307400443, 16O 15.99491461957 and 32S 31.9720711744. [1] Average mass uses standard atomic weights instead and is always somewhat higher. Expasy's PeptideMass tool returns either average or monoisotopic values, so you can cross-check your arithmetic and must state which one you used. [3]

Worked example with a fictional sequence, GAVFK. Residue masses: G 57.02146, A 71.03711, V 99.06841, F 147.06841, K 128.09496. Their sum is 502.29035; adding water (18.01056) gives a neutral monoisotopic mass M of 520.3009 for the formula C25H40N6O6. Modifications change this number: an amidated C-terminus, an acetylated N-terminus, a disulfide bond or a salt form must be reflected in the expected mass, so copy the exact specification, not just the one-letter sequence.

Charge states: why one peptide shows several peaks

Electrospray ionization often puts more than one proton on a peptide, so the same molecule appears at several m/z values. Agilent's deconvolution documentation describes the logic: a set of peaks is treated as one component when consecutive charge states give the same molecular weight, and software converts the multiply charged spectrum into a neutral-mass spectrum. [4] The proton mass used for that conversion is 1.007276466 u (CODATA 2022). [2]

To check a report by hand, take each listed peak, assume a charge n, and compute neutral mass = n x (m/z) - n x 1.007276. In the GAVFK example, 261.1577 x 2 - 2.014553 = 520.3009, the same neutral mass as the singly charged peak at 521.3082. If the certificate only shows a deconvoluted mass, ask for the raw spectrum or at least the ion list. A deconvoluted number with no spectrum cannot be checked.

Tolerance: how close is close enough

Mass error is usually expressed in parts per million: (observed - theoretical) / theoretical x 1,000,000. If a report lists [M+H]+ observed at 521.3075 against a theoretical 521.3082, the error is -1.3 ppm. If it lists 521.3 with no decimals beyond one place, it is reporting at a much coarser resolution, and the acceptable window should be correspondingly wider.

Laboratories price and report these as different services. Ohio State's core, for example, lists accurate-mass molecular weight determination (ESI or MALDI) separately from nominal-mass MALDI. [5] A nominal-mass result is still useful, but it cannot separate candidates that differ by less than about one mass unit. The certificate should state its acceptance window. If it does not, ask the issuing laboratory what tolerance it applied and whether the value is monoisotopic or average.

What mass spectrometry does and does not prove

A mass match is evidence that a molecule of that mass was present in the sample tested. It is not evidence about how much of it there is or how pure the material is. Two sequences with the same amino acids in a different order have exactly the same mass: GAVFK and KFVAG both compute to 520.3009. Swapping an L-amino acid for its D-form also leaves the mass unchanged. A deletion is easier to spot: AVFK, missing the glycine, computes to 463.2794, a 57.0215 drop.

A five-minute check before filing the certificate

If the report has no spectrum, no ion type and no expected value, file it as unresolved and ask the issuing laboratory for the underlying data. This review covers documentation only. It says nothing about suitability for any use outside the laboratory.

• Copy the exact sequence and modifications from the order, not from the product name.
• Compute the monoisotopic neutral mass yourself or with Expasy PeptideMass, and note which mass type you used. [3]
• Convert it to every ion the report lists, using 1.007276 per proton. [2]
• Calculate the ppm error for each listed ion and compare it with the tolerance printed on the report.
• Confirm the lot number on the MS page is the lot on your vial and on the HPLC page.
• Record the result as identity supported, not supported, or unresolved. Do not record it as purity.