Mass spectrometry (revision 37)

Mass spectrometry measures the mass-to-charge ratio of ionised molecules. For peptides it is the ordinary means of confirming identity: an observed molecular mass agreeing with the calculated mass excludes most alternatives, and fragmentation can confirm the sequence itself.^[1]

A purity determination by chromatography establishes that one species dominates a sample; it does not establish which species. These are separate questions and require separate determinations. A certificate reporting purity but no identity leaves the more fundamental of the two unanswered.^[2]

Peptides ionised by electrospray carry multiple charges, so a single peptide produces a series of peaks at different charge states. The instrument software deconvolves this envelope into one neutral mass, and it is that deconvolved mass that appears on a report.^[1][3]

Electrospray disperses a solution into charged droplets in an electric field; solvent evaporates and ions are released into the gas phase. It is a soft technique — the molecule survives largely intact — which is why it suits peptides and proteins. Matrix-assisted laser desorption is the other common soft method and tends to produce singly charged ions.^[1]

Mass analysers differ in resolution and in mass accuracy. A quadrupole gives unit resolution and is adequate for confirming a mass to within a dalton; time-of-flight and orbital-trap analysers give sufficient resolution to determine a mass to a few parts per million, which distinguishes species a quadrupole cannot.

The distinction matters for peptide work because the confounders are close in mass. Deamidation adds 0.984 Da, which unit resolution cannot resolve from the parent; oxidation adds 16 Da, which it can. A report stating an observed mass without stating the resolution of the instrument understates the ambiguity.^[2]

Intact-mass agreement is a strong but incomplete identification. Two different sequences composed of the same residues have identical masses, and inverting the order of two residues changes nothing about the intact mass. Only fragmentation — tandem mass spectrometry, which selects a precursor ion, breaks it and measures the fragments — addresses sequence order.^[1]

Stereochemistry is invisible to mass spectrometry entirely. A D-amino acid substitution is isobaric with the L form and requires chiral analysis to detect. For peptides synthesised with racemisation-prone residues this is a real rather than theoretical gap.

Mass spectrometry is not inherently quantitative. Ionisation efficiency varies by orders of magnitude between molecules and is affected by the matrix, so peak intensity does not translate into concentration without calibration against a standard of the same substance.^[2]

Where quantification is required, the usual approach couples chromatography to mass spectrometry — LC-MS — and calibrates against a reference standard, ideally with an isotopically labelled internal standard. This is standard practice in bioanalysis and is not typical of the certificates seen in research-chemical supply; a quantitative claim requires a validated procedure behind it.^[4]

The practical consequence is that a mass spectrum on a certificate answers "is this the right molecule" and nothing else. It does not report purity, does not report content, and does not establish that minor species absent from the spectrum are absent from the material — poorly ionising impurities may simply not appear.^[1]

• Electrospray ionisation
• Liquid chromatography-mass spectrometry
• Deamidation
• Methionine oxidation
• Amino acid analysis
• Reference standard