Identity by mass spectrometry

The sample is separated chromatographically and the eluent passed into an electrospray source, where the analyte is ionised at atmospheric pressure and enters the mass analyser as a population of multiply charged ions. A 4 kDa peptide typically appears as a series of charge states from roughly 3+ to 6+, each at a different mass-to-charge ratio.

Deconvolution collapses that series back to a single neutral mass. Because the charge states are mutually constraining, the deconvoluted figure is far more reliable than any single peak, and a spectrum that fails to deconvolute cleanly is itself diagnostic: it usually means co-elution or adduct formation that needs resolving before a number is reported.

Acceptance is agreement between the measured and theoretical monoisotopic or average mass. For synthetic peptides the practical criterion is a difference under one dalton, and in a well-behaved system under 0.5 Da. Larger proteins are quoted against average mass because the isotope envelope is no longer resolved.

Reversed-phase retention is governed by hydrophobicity. Two peptides with different sequences but similar hydrophobic character elute at similar times, and a single-point retention match therefore carries far less information than it appears to.

Three cases recur. Deletion sequences missing a small residue shift retention only slightly. Diastereomers arising from racemisation during synthesis may not separate at all under standard conditions. Structural analogues sold as the labelled compound are routinely close enough in retention to pass a visual comparison against a reference chromatogram.

Mass resolves the first and third of those directly. The second requires tandem MS or a chiral method, and where a determination cannot resolve a question, that limitation belongs on the certificate rather than in a footnote.

Intact mass confirms composition, not order. Two peptides built from the same residues in a different sequence share a mass. Where that ambiguity is material, the precursor is isolated and fragmented, and the resulting b and y ion series read the sequence directly.

Leucine and isoleucine remain indistinguishable by ordinary collision-induced dissociation because they are isomeric; that is a property of the physics, not a shortcoming of a particular laboratory, and any report implying otherwise should be treated with suspicion.

Identity work depends on holding a characterised reference standard for the target. Without one there is a mass but no anchor, and the analysis reduces to a plausibility argument.

Where no standard is held for a compound, the honest response is to decline the determination rather than to report a mass and let the reader assume it was compared with something. ANALYTON states the standard used, its source and its lot on the certificate.