What is Mass spectrometry?
Also called: MS, Mass spec, LC-MS, ESI-MS
An analytical technique that ionises molecules and measures their mass-to-charge ratio (m/z); for peptides it confirms identity by matching measured mass to the mass calculated from the sequence.
By the APL Research Team · Updated
Mass spectrometry (MS) converts molecules into gas-phase ions and sorts them by mass-to-charge ratio, m/z. For a synthetic peptide it answers the question HPLC cannot: is the main component the sequence that was ordered? Agreement between the observed mass and the mass calculated from the sequence is the usual identity criterion on a certificate of analysis.
Ionisation and charge states
Peptides are usually ionised by electrospray (ESI), often straight from an HPLC column (LC-MS), or by MALDI. Electrospray adds protons, and larger molecules collect several. The spectrum therefore shows a run of peaks for one molecule, each differing from its neighbour by a single charge, the signature pattern described in the foundational electrospray work [1]. Each peak sits at m/z = (M + n × 1.00728) ÷ n, where M is the neutral mass and 1.00728 Da is the mass of a proton.
Semaglutide (C187H291N45O59) illustrates the effect:
| Species | Monoisotopic value |
|---|---|
| Neutral molecule (M) | 4111.12 Da |
| [M+3H]³⁺ | m/z 1371.38 |
| [M+4H]⁴⁺ | m/z 1028.79 |
| [M+5H]⁵⁺ | m/z 823.23 |
A 4.1 kDa peptide thus appears near m/z 1029 and 823, not near 4111. Software deconvolutes the charge series back to one neutral mass, and that deconvoluted figure is what most COAs report.
Monoisotopic, most abundant or average?
About 1.1% of natural carbon is carbon-13, so every ion appears as a cluster of isotope peaks spaced 1/n apart on the m/z axis. With 187 carbons, semaglutide's monoisotopic peak (no heavy isotopes) is only about 40% as tall as the tallest peak in its cluster, which carries two. Its average molecular weight, the figure used for weighing, is 4113.6 g/mol. A COA quoting 4113.6 and a spectrum labelled 4111.1 can both be right; before treating a result as "2 Da off", check which convention each number uses.
What MS catches
Most synthesis-related impurities, including deletion sequences, residual protecting groups and oxidised side chains, change the mass [2]:
| Change | Mass shift (monoisotopic) |
|---|---|
| Missing glycine | −57.02 Da |
| Missing lysine | −128.09 Da |
| Methionine oxidised to sulfoxide | +15.99 Da |
| Asn or Gln deamidated | +0.98 Da |
| tert-Butyl protecting group left on | +56.06 Da |
| Racemisation (L → D) or isoaspartate | 0 Da |
What it misses
The last row is the blind spot. Diastereomers produced by racemisation have exactly the target's mass [2], so separating them needs chromatography. MS is also not a purity test: ionisation efficiency differs from molecule to molecule, so relative peak heights in a spectrum do not translate into percentages. Tandem MS (MS/MS), which breaks the ion along its peptide bonds, goes a step further and confirms the order of residues rather than just their total.
Checking a result
The peptide molecular weight calculator gives average and monoisotopic masses and the [M+H]⁺ value for any sequence, including acetylated, amidated and disulfide-bridged forms, so a reported mass can be checked independently. Mass values for larger peptides are easier to interpret with the dalton and charge-state arithmetic above in mind. The COA guide covers how identity results are presented alongside purity.
References
- 1.Fenn JB, Mann M, Meng CK, et al. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989. PubMed 2675315
- 2.D'Hondt M, Bracke N, Taevernier L, et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014. PubMed 25044089