Mass spectrometry and peptide characterisation

Ionisation: ESI and MALDI

Two ionisation techniques dominate peptide work. Electrospray ionisation generates ions from a liquid stream, which makes it straightforward to couple directly to an HPLC system so that separation and identification happen in one run.

Matrix-assisted laser desorption/ionisation embeds the sample in a crystalline matrix and desorbs it with a laser pulse. It tolerates salts and buffers better than electrospray and is often more convenient for rapid checks on solid samples.

The two produce different charge behaviour. Electrospray typically generates multiply charged ions, so a single peptide appears as a series of peaks at different charge states. MALDI predominantly generates singly charged ions, giving a simpler spectrum.

Charge states and m/z

A mass spectrometer measures mass-to-charge ratio, not mass. For a singly charged ion these are numerically close, but for multiply charged ions they are not, and the distinction is where most misreadings originate.

An electrospray spectrum of a peptide of around 3600 Da might show ions at roughly m/z 1201 for the triply charged species and m/z 901 for the quadruply charged species. Neither number is the molecular weight. Deconvolution software combines the charge-state envelope to produce a single neutral mass, and it is that deconvoluted value which should be compared against the theoretical mass.

Average mass and monoisotopic mass

Every element occurs as a mixture of isotopes. Average mass uses the natural abundance-weighted mean atomic mass of each element; monoisotopic mass uses the mass of the most abundant isotope of each element.

For small peptides the two differ by roughly one to two daltons; for larger molecules the gap widens. Comparing an observed monoisotopic mass against a theoretical average mass, or the reverse, produces an apparent discrepancy that is purely an artefact of the comparison.

A certificate should state which convention it uses. Where it does not, the size of the mismatch usually reveals it.

Diagnostic mass differences

Because modifications and degradation products shift mass by characteristic increments, the difference between an observed and an expected mass is often directly interpretable.

• +16 Da — oxidation, most commonly of methionine to the sulfoxide.
• +1 Da — deamidation of asparagine or glutamine.
• −0.98 Da relative to the free acid — C-terminal amidation.
• +42 Da — acetylation, frequently at the N-terminus.
• −18 Da — loss of water, which can indicate dehydration or cyclisation.
• −2 Da — formation of a disulfide bond between two cysteine residues.
• A shortfall matching a residue mass — a deletion sequence missing that residue.

What intact mass does not establish

An intact mass measurement confirms that the total composition is consistent with the intended sequence. It does not confirm the order of the residues.

Two peptides containing the same amino acids in a different order have identical intact masses and are indistinguishable by that measurement alone. Distinguishing them requires tandem mass spectrometry, in which the peptide is fragmented and the resulting fragment masses are used to read the sequence.

For routine confirmation of a well-characterised synthetic peptide, intact mass plus chromatographic purity is generally the standard combination. Sequence-level confirmation is the stronger evidence where the identity itself is in question.

Frequently asked questions

What is the difference between average and monoisotopic mass?

Average mass uses the natural abundance-weighted mean atomic mass of each element; monoisotopic mass uses the most abundant isotope of each element. They differ by roughly one to two daltons for small peptides, and the gap widens with molecular size. Comparing one against the other creates an artificial discrepancy.

Why does one peptide produce several peaks in an ESI spectrum?

Electrospray ionisation typically produces multiply charged ions, so a single peptide appears as a series of peaks at different charge states. Deconvolution combines that envelope into a single neutral mass, which is the value to compare against the theoretical mass.

Does a correct intact mass prove the sequence is correct?

No. Intact mass confirms overall composition, not residue order. Two peptides with the same amino acids in a different order have the same intact mass. Confirming sequence requires tandem mass spectrometry with fragmentation.

Sources and further reference

• Domon B., Aebersold R. Mass spectrometry and protein analysis. Science, 2006 (PMID 16614208)
• PubChem CID 146675088 — MOTS-c
• Towards a Consensus for the Analysis and Exchange of TFA as a Counterion in Synthetic Peptides. Pharmaceuticals, 2025 (PMID 40872554)

Compounds this applies to

The considerations above are not abstract. Each of these compounds is listed in the Peptide Tactics catalog for research use in India, with its reference data, analytical notes and documentation status set out in full.

See the full research peptide catalog, the India sourcing guide or how to request documentation.

Continue reading

• HPLC analysis of peptides — How reversed-phase HPLC separates peptides, which method parameters change the reported purity, and how to read a peptide chromatogram critically.
• Peptide purity explained — What a peptide purity percentage actually measures, why it differs from net peptide content, and which method details make a purity claim readable.
• Understanding peptide certificates of analysis — How to read a peptide certificate of analysis: which fields carry real information, how to spot a template, and what a COA cannot tell you.