[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.

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.

post #2 answers the question as asked. The question underneath it is different.

Purity and identity are different questions: LC-MS establishes that the species at a retention time has the expected mass. It does not establish how much of the sample is that species (that is what LC-UV purity answers).

Electrospray ionisation produces multiply charged ions. For a 4 kDa peptide you expect mostly 2+, 3+, and 4+ charge states. Reading an electrospray spectrum means recognizing the envelope, not looking for one peak.

Calibration matters: a high-resolution instrument out of calibration can report mass with ppm error large enough to be uninformative. Check when the instrument was last calibrated before trusting the reported accuracy.

I read post #4 twice before replying, because I had assumed the opposite.

Resolution: "high resolution" commonly means <5 ppm across the mass range. Unit-resolution instruments achieve ±1 Da at best and cannot distinguish two species differing by less than 1 Da in total mass.

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.

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.

Picking up post #6: that is the part I would want checked first.

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.

What mass accuracy establishes: the measured mass is consistent with a specific composition. What it does not establish: purity, sequence order, stereochemistry, or the absence of an isobaric species. Every one of those requires something else.

On post #7 — agreed on the reasoning, with one qualification.

Practical note that does not fit anywhere else. Whatever you conclude from this topic, write down what you did and when. The single most useful thing in your own records is not any individual result; it is that they are dated and consecutive.

post #11 answers the question as asked. The question underneath it is different.

Mass accuracy is expressed in parts per million. It is the difference between observed and theoretical mass divided by theoretical mass, multiplied by a million. A high-resolution instrument in good calibration achieves low single-digit ppm on a peptide of this size.

Charge states observed: for semaglutide (4113.6 Da) the doubly charged ion appears at m/z ≈ 2057, triply charged at ≈ 1371, quadruply at ≈ 1029. Those are the positions to look for; the heights depend on the ionization efficiency.

Common adducts: sodium adds ≈22, potassium adds ≈38 compared to hydrogen. A [M+Na]+ peak is common and its mass is predictable from the base mass.