Mass spectrometry (revision 17)

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.

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