Deletion sequences: expected mass shifts by residue — the long version

On the subject in the title: Deletion sequences: expected mass shifts by residue — the long version Working notes rather than a conclusion.

Working through the identity arithmetic and I would like it checked.

semaglutide has a monoisotopic mass close to 4113.6 Da. On an electrospray instrument I would expect to see the multiply charged series rather than the intact singly charged ion, so for the doubly charged species I calculate (4113.6 + 2 x 1.00728) / 2, and for the triply charged the analogous expression.

The observed values in the report sit within a few ppm of those. My question is what that actually establishes, because I have seen people treat a mass match as a purity result and I do not think it is one.

Truncation products: fragments from incomplete synthesis or from degradation. They elute quite differently from the intact peptide because they are much smaller and have different hydrophobicity. They are usually well separated.

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.

Worth separating two things that post #3 runs together.

Two things before anyone answers the substance.

First, the context in the first post is clear and specific. Second, the question is framed so that an answer can actually address it. Both are the norm here and both matter more than they sound.

Picking up post #2: that is the part I would want checked first.

Oxidation at methionine and tryptophan: adds 16 per oxygen. Usually elutes earlier. Oxidation is common in storage, especially if the solution is exposed to light or if antioxidants are not present.

Coming back to post #4, because the follow-up matters more than the original answer.

Deamidation at asparagine and glutamine: adds 1 approximately. Frequently appears as a close-eluting pair. It is a chemical modification that occurs during storage.

For anyone arriving from a search: the marked solution above is the direct answer, and the replies underneath it add the caveats that make it safe to use.

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.

Incomplete deprotection: mass higher by the protecting group mass. Usually markedly later eluting. A synthesis artifact from incomplete removal of protecting groups.

Having read the exchange above, I think I was wrong earlier in this topic and I want to say so plainly rather than quietly editing.

The correction was fair and I had been repeating something I had not checked carefully enough.

Acetate content: counter-ion content. Trifluoroacetate or acetate from the salt form of the peptide. Affects mass calculations and should be stated on a complete certificate.

post #11 is right about the mechanism and I think understates the practical bit.

Residual solvents: traces of solvents used in purification. These are usually tested by gas chromatography, not by HPLC. A specification for residual solvents should be stated separately from the purity.

I disagree with the reply above, and I think the disagreement is substantive rather than terminological.

The distinction being drawn does not survive when you look at the published data for this specific question. I would be glad to be shown wrong on this, because the version I am arguing against is more convenient.