[2026 update] Charge states for a 4 kDa peptide, worked through

On the subject in the title: Charge states for a 4 kDa peptide, worked through 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.

Helpful, and short, which on this subject is harder than long.

Adding the measurement that the opening post says would settle it.

Sample matrix effects: if a sample is dissolved in a complex matrix, other compounds in the matrix can suppress the peptide signal. Clean samples give higher sensitivity than dirty samples.

I keep a log of this specifically because memory is unreliable about it.

Desalting before analysis: some samples need desalting to remove salts that suppress the peptide signal. Report whether desalting was used, because it can affect the apparent ionization efficiency and the reported purity.

One case, stated as one case.

Practical experience of Charge states, offered as one case with the conditions stated, not as a general finding. Conditions first, because they are what make it interpretable.

I had written a reply contradicting post #4 and deleted it. Here is what survived.

I would be cautious about generalising from the Charge states example above. It is a good example. It is one example.

Positional isomers and epimers are mass-identical. Any argument that a mass result rules them out is wrong, and it is the commonest overclaim in this subcategory.

The short answer was in the first line; everything after is the working.

Trifluoroacetate adducts are common in material purified with TFA and are one reason a mass spectrum from a peptide can look busier than expected.

I have seen it go both ways, which is why I hedge.

Fine by me. I had wanted a stronger conclusion and there is not one available.

Charge states was covered in the wiki last year and the page has a review date on it, which is a better starting point than my memory of a thread.

Worth separating two things that post #7 runs together.

An honest declaration on Charge states: I have a prior here and it is strong enough that you should weight what I say downward. Stating it rather than hiding it.

This follows post #11 rather than contradicting it.

The arithmetic for a doubly charged species is (M + 2 x 1.00728) / 2, and the analogous expression for higher charge states. Working it through once makes the reported values legible.

That is the version I use. It may not be the version that is correct.

Where I would push back on the Charge states consensus is the confidence, not the direction. The direction looks right. The confidence is borrowed.

Small methodological point on Charge states: repeating a measurement is cheap and resolves most of what is being argued about here at no cost to anyone.