[2026 update] Charge states for a 4 kDa peptide, worked through posts 31–60

The arithmetic in post #43 is right; the assumption feeding it is the part to check.

Electrospray on a peptide of this size gives a multiply charged series rather than a single ion. Seeing only one charge state usually means the deconvolution has already been done for you, which is worth knowing.

That is one dataset and I would not build a rule on it.

Answering the question post #44 raises rather than the one it answers.

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.

Worth reading the earlier posts in this thread before acting on mine.

The most useful thing anyone has posted about Charge states in this category was a table of what had been measured and by whom. That is what I would want again.

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.

Seconded. It reads as careful rather than confident, which is the right register.

Adding a null result on Charge states. I looked, carefully, and found nothing, and null results deserve posting precisely because they never are.

Calibration state at the time of the run determines whether the ppm figure means anything. A report that states when the instrument was last calibrated is unusual and is worth more than one that does not.

Noting that the question and the thing people usually mean by it are different.

This follows post #51 rather than contradicting it.

What I want from this Charge states thread is the list of things that would need to be true for the claim to hold. If we can write that list, we can check it.

Coming back to post #51, because the follow-up matters more than the original answer.

Adding a small correction to the Charge states summary above rather than a disagreement with it. The substance holds; one of the figures is out by a factor that matters.

An acylated peptide has a mass that reflects the modification, so comparing against the mass of the unmodified backbone gives a mismatch that is not an error.

Written quickly, so the reasoning may be tighter than the wording.

Picking up post #54: that is the part I would want checked first.

Quantitation by MS: most quantitation is done by LC-UV detection at 214 nm, not by MS, because extinction coefficients are better known. MS can quantify if an internal standard is used but that requires preparation.

Not a conclusion. A place to stand while looking for one.

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

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.

Noted, and I have changed what I was going to do on the strength of it.

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.