D-amino acids are invisible to mass spectrometry, and that is the whole problem

The useful way to think about a mass spectrometer is as an instrument that answers one question extremely well and is silent on several adjacent ones. Deamidation of an asparagine residue converts an amide to a carboxylic acid and adds nine hundred and eighty-four thousandths of a dalton. Oxidation of a methionine adds one oxygen atom, fifteen point nine nine five daltons. Inversion of a single stereocentre from the L to the D configuration adds nothing whatever. Those three numbers — roughly one, roughly sixteen, and zero — describe the three most consequential things that go wrong with a synthetic peptide, and an instrument’s ability to detect them falls off in exactly that order.

Electrospray, and why it produces a family of ions

Electrospray ionisation works by pumping a solution of the analyte through a fine capillary held at a potential of a few kilovolts relative to the instrument’s entrance. The liquid emerging from the tip forms a cone and then a jet of charged droplets. As solvent evaporates the droplets shrink, the charge density on their surfaces rises, and at the point where electrostatic repulsion exceeds surface tension they fission into smaller droplets. Repeat this enough times and what is left is a bare, charged analyte ion in the gas phase.

Because the charge is acquired in solution and retained through desolvation, a peptide with several basic residues will carry several protons, and the population of ions reaching the analyser is distributed across charge states. This is the defining characteristic of electrospray spectra and the reason they look bewildering to a first-time reader: a single pure compound produces four or five prominent peaks, none of them at the molecular weight.

The distribution is not noise. It carries information about the number of accessible basic sites and about the conformational state of the molecule in solution, and it shifts predictably with mobile-phase composition and pH. It also has a practical advantage that matters for identity work: dividing the mass by three or four brings a large peptide into the range where instruments achieve their best resolving power and accuracy.¹

Isobaric and near-isobaric substitutions

Two species are isobaric if their masses are identical to the precision of the measurement, and the term covers two quite different situations. True isobars have identical elemental compositions: leucine and isoleucine are structural isomers of one another, as are the aspartate and isoaspartate products of deamidation, and no mass measurement at any resolving power will separate them. Near-isobars have different compositions that happen to give similar masses, and these are resolvable given sufficient performance.

The canonical near-isobaric pair in peptide work is glutamine against lysine, differing by 0.036 daltons — nine parts per million on a four-thousand-dalton peptide, and therefore a discrimination that requires an orbital trap or better. A second is the classic composition ambiguity in which a combination of light elements substitutes for a heavier one at nearly the same nominal mass; the mass defect of hydrogen relative to the heavier elements is what makes these separable at high resolving power and indistinguishable at low.²

The reason this matters commercially is narrow but real. A synthesis error that substitutes one residue for another may be invisible on a low-resolution instrument, present at a few per cent, and chromatographically unresolved from the parent under a fast gradient. The combination of a twelve-minute purity method and a unit-resolution identity check is not a conspiracy; it is simply a pair of tests neither of which is looking in that direction.

Deamidation: 0.98 daltons, and where it hides

Deamidation of asparagine proceeds through a five-membered succinimide intermediate formed by nucleophilic attack of the following residue’s backbone nitrogen on the asparagine side-chain carbonyl. Hydrolysis of the intermediate yields aspartate or isoaspartate, in a ratio typically favouring the isoaspartate form. Glutamine deamidates by an analogous but slower route. The rate depends strongly on pH, temperature, and the identity of the residue immediately following the asparagine, with glycine and serine accelerating it markedly.³

The analytical difficulty is threefold. The mass increase is 0.984 daltons, which requires only modest resolving power to see at low molecular weight and becomes demanding as the peptide gets larger. The aspartate and isoaspartate products are exactly isobaric with one another, so distinguishing them requires either a chromatographic separation that happens to resolve them or a specific enzymatic assay. And deamidated species often elute close to the parent under reversed-phase conditions, so a fast gradient may not separate them either.

The result is a degradation product that is common, that has real consequences for biological activity, that accumulates in storage, and that a certificate produced by a unit-resolution instrument on a twelve-minute gradient is structurally unable to detect. When the Journal describes a certificate as silent on stability, this is a large part of what is meant.

Oxidation: sixteen daltons, and the one that gets caught

Methionine oxidises to the sulphoxide with a mass increase of 15.995 daltons, and on to the sulphone at a further 15.995. Tryptophan and histidine oxidise by related routes. The chemistry is driven by dissolved oxygen, by trace peroxides in excipients and in some grades of polysorbate, by light, and by transition-metal contamination, and it proceeds in lyophilised material as well as in solution, though more slowly.

Analytically this is the easy case, and it deserves to be described as such in an article otherwise concerned with what cannot be seen. A sixteen-dalton shift is resolvable on essentially any instrument, and the oxidised species is usually chromatographically distinct enough to appear as a separate peak under a reasonably shallow gradient. A spectrum showing a plus-sixteen satellite at a few per cent of the parent intensity is unambiguous evidence of oxidation, and its absence is meaningful evidence of the opposite.

Which is why the Journal’s standing request to laboratories in this market is for the spectrum rather than the verdict. A reproduced spectrum, even at the modest resolution of a routine instrument, allows a reader to look for the plus-sixteen satellite themselves. A conformance statement does not, and cannot be made to. This is the cheapest available improvement to identity reporting in the trade and it consists of printing a picture the laboratory has already produced.

The inversion that weighs nothing

Amino acids other than glycine are chiral, and peptide synthesis is performed with L-configured building blocks. Racemisation during synthesis — most commonly at cysteine, histidine and aspartate residues, and promoted by prolonged base exposure during coupling and deprotection — produces a peptide containing one or more D residues. The resulting molecule has the same elemental composition, the same monoisotopic mass, the same average mass, and the same fragmentation masses as the intended product.

Mass spectrometry cannot detect it. This is not a limitation of any particular instrument; it is a consequence of what the technique measures. Reversed-phase chromatography sometimes separates diastereomeric peptides, and where it does the epimer appears as a shoulder or a satellite peak of unassigned identity — which is one reason a chromatogram with an unexplained minor peak deserves more attention than a purity percentage does. Where the epimer co-elutes, no routine analysis in this market would find it.

Deliberate detection requires chiral amino-acid analysis after total hydrolysis, or digestion with a stereospecific protease that fails to cleave across a D residue, or in some cases ion-mobility separation. None of these is offered as a standard service to private buyers by any of the four testing services this market relies on, and the Journal’s position is that this is a genuine gap rather than a failing on their part: nobody has ever been asked to price it.

The standing rule in this department

This publication applies one rule to every identity claim it reports, and it is worth stating in isolation because it governs the rest. A mass measurement supports a statement about composition. Only a fragmentation or mapping experiment supports a statement about sequence. Where a source says identity was confirmed, we report that a mass was measured, unless we have seen evidence of the second kind.

The rule has consequences we accept. It makes our coverage read as more sceptical than the underlying documents, because the documents claim more than they establish. It occasionally irritates laboratories which have in fact done sequence-level work and have simply not printed it, and the remedy there is a two-line email which we are glad to receive. And it means we cannot describe any research-grade vial in this market as sequence-confirmed, because on the evidence available to us almost none are.

What the rule is not is an accusation. Nothing in this article suggests that vendors are selling material other than what they label, and the Journal has no evidence of that in respect of any company it covers. The claim is narrower and, we think, harder to argue with: the documentation in general circulation does not have the discriminating power that the language on it implies, and the gap between the two is where every avoidable dispute in this market begins.

If this market spent one more pound on identity, where should it go

A fair question, and the Journal’s answer has changed. Our first instinct was to argue for sequence confirmation on every lot, and the arithmetic does not support it: peptide mapping on every batch would raise the analytical cost per vial by a multiple, and the failure mode it protects against — a wholly substituted or permuted sequence — is not the one we see evidence of.

The better allocation, on our present assessment, is orthogonal. Identity by high-resolution intact mass on every lot, at a resolving power sufficient to resolve a one-dalton shift at the parent mass, with the spectrum reproduced. Sequence confirmation once per synthesis campaign rather than once per lot, on the reasoning that the sequence is a property of the process and the lot-to-lot risk is degradation rather than misconstruction. And a chromatographic method shallow enough to separate the deamidated form, because that is the change most likely to have occurred between the certificate and the buyer.

That package is not expensive. Two of the twenty companies in our dossier programme already do something close to the first item, and one has told us it is costed for the second. Whether any of it happens depends on whether buyers ever ask, which is a market question rather than a scientific one and is therefore the harder of the two.

Readers who take one thing from this piece should take the arithmetic. Isotope spacing is one over the charge. Deamidation is one dalton, oxidation is sixteen, and stereochemical inversion is nothing at all. A tolerance of ±1 dalton on a four-thousand-dalton peptide is two hundred and forty parts per million and excludes almost nothing worth excluding. Those four facts are sufficient to read most of the identity claims in circulation, and they fit on the back of an envelope.