On the subject in the title: Deletion sequences: expected mass shifts by residue — the long version Working notes rather than a conclusion.
Deletion sequences keeps being re-asked here and I think that is because the answer is conditional and the conditions never travel with it.
Attempting to write the conditional version down properly. Corrections welcome and expected — I would like this to end up as a maintained page rather than a fifth thread.
Saving this. It is the version I will quote when the question comes round again.
Off-target structures: if the sequence synthesis goes wrong, a completely different amino acid can be incorporated. The resulting off-target peptide is a structural isomer with the same mass but a different sequence. No chromatographic purity method detects this without a reference standard.
A single observation, in a thread that deserves better than single observations.
The opening post put the caveat in the right place and I want to underline it.
The confident answers on Deletion sequences and the well-sourced answers are not the same answers, which is the most useful thing I have learned reading this category.
I read post #4 twice before replying, because I had assumed the opposite.
Dimer and higher-order multimers: two or more peptide molecules bonded together. They appear at double the mass and higher. They may or may not separate from the monomer on HPLC depending on the method.
Scavengers and cleavage-cocktail residues can persist and appear as small early-eluting peaks. They are process-related rather than sequence-related, which is a useful distinction when reading a profile.
Worth reading the earlier posts in this thread before acting on mine.
That is the distinction I keep failing to hold on to. Written down now.
Post #11 describes the usual case. This is about the unusual one.
Incomplete deprotection leaves a protecting group attached, raising the mass substantially and usually pushing retention much later. A late-eluting peak on a peptide chromatogram is worth asking about.
Take it as a starting point and not as a specification.
Deletion sequences arise from incomplete coupling and are lower by one residue mass. Where they elute depends on the hydrophobicity of the residue that is missing, so they can appear on either side of the main peak.
Someone should write this up properly, and it should probably not be me.
Post #15 is the version of this I will quote in future. One addition.
Deletion sequences (incomplete coupling during synthesis): lower in mass by one residue. Chromatographically they usually elute earlier or later depending on the residue's hydrophobicity. They are the most common impurity in solid-phase synthesis.
One more caveat and then I will stop qualifying: the sample selected itself.
The documentation on Deletion sequences is better than this thread and I say that as someone who has posted in the thread.
Clear enough that I do not think I have a follow-up, which is unusual.
Peptide impurities that differ by a single residue are the hardest to resolve and the most likely to be biologically relevant, which is an unfortunate combination.
One case, stated as one case.
Oxidation at methionine or tryptophan adds sixteen per oxygen and typically elutes earlier. In aged material it is the modification that grows.
I would be glad to be shown a cleaner way of putting this.
Trifluoroacetate is a counter-ion rather than an impurity, and it appears in the mass balance rather than in the chromatogram. Conflating the two accounts for several confused threads here.
The right answer here may simply be that it has not been measured.
Narrowing post #20, because the general version has more than one answer.
Relative response factors mean impurities are not detected in proportion to how much of them is present. A one per cent peak is not one per cent by mass unless the response factors happen to match.
No disagreement from me. Posting only so the question does not look ignored.
Incomplete deprotection: mass higher by the protecting group mass. Usually markedly later eluting. A synthesis artifact from incomplete removal of protecting groups.
Aggregates: multiples of the monomer mass. May not elute at all under a standard reversed-phase method. A species that does not come off the column does not appear in the area percentage.
That is one dataset and I would not build a rule on it.
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