Charge states for a 4 kDa peptide, worked through

Charge states has a well-known answer and a correct answer, and the interesting work is establishing that they are the same. Nobody has done that here yet.

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.

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.

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

Picking up post #15: that is the part I would want checked first.

Something worth flagging about charge states: the strongest-sounding claims in this thread are the ones with no source attached, which is the usual pattern and not a coincidence.

Post #15 put the caveat in the right place and I want to underline it.

An observation about charge states that I cannot explain and am posting anyway, on the principle that unexplained observations are more useful public than private.

Building on post #19 rather than restating it.

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.

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.

Post #18 and I disagree about the size of the effect, not about the direction.

Marking my uncertainty on charge states explicitly. I am confident about the direction, much less confident about the size, and not confident at all that it generalises past the case in the first post.

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.

This follows post #24 rather than contradicting it.

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.

I looked this up rather than remembered it, which is the right order.

Worth separating two things that post #22 runs together.

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.

That is what I would do. It may not be what is correct.

Nobody has said the unglamorous part of charge states yet, so: most of the variation is explained by things that are boring to write about and easy to check.

My experience of charge states contradicts the reply above. I am posting it as a data point rather than as a refutation, because one person's experience is exactly that.

Picking up post #28: that is the part I would want checked first.

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.

Small point, but it is the one that usually catches people.

Genuine question rather than a rhetorical one: has anyone here actually observed charge states, as opposed to read about it? The thread is long and I cannot tell.