This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1 · go to the accepted answer.
I read post #29 twice before replying, because I had assumed the opposite.
Where the charge states reasoning breaks down for me is the step from the group result to the individual case. That step is almost never argued for.
Post #29 answers the question as asked. The question underneath it is different.
Resolution and mass accuracy are different specifications. An instrument can resolve two species and still assign their masses imprecisely, and the reverse is also possible.
If anyone can point at the primary source I would be grateful.
Trifluoroacetate adducts are common in material purified with TFA and are one reason a mass spectrum from a peptide can look busier than expected.
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.
Everything in post #33 holds. The case it does not cover is the one I have.
I would rather this thread reach "we do not know" about charge states than reach a confident answer that nobody can support when asked.
Thank you for the correction. I would rather find out here than later.
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.
Trying to state the charge states position in a way that someone who disagrees would recognise as fair, because I do not think the version in this thread passes that test.
One caution on charge states: everything above assumes the underlying documentation is what it claims to be. That assumption is doing real work and is rarely stated.
The honest summary of what a mass result buys you: it narrows the field of what the material could be, considerably. It never closes it, and no certificate should be read as though it had.
Everything in post #40 holds. The case it does not cover is the one I have.
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.
That is my reading. Someone else read the same page differently and was reasonable.
This follows post #42 rather than contradicting it.
Reading this charge states thread as someone who came in with a fixed view: the third and seventh replies moved me and the confident ones did not.
Right — I had this wrong and I am glad to have read it before it mattered.
Whatever the answer on charge states turns out to be, the method for getting there is the same: state the assumption, do the arithmetic in public, invite the correction.
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 am describing what is, rather than arguing for what should be.
Where I part company with post #48, and it is a narrow parting.
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.
I would call that likely rather than established.
Bookmarking this. I will come back when I have something worth adding.
Answering the question post #51 raises rather than the one it answers.
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.
I checked the source rather than the summary, and they differ.
The arithmetic in post #51 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 the honest state of it as of this week.
Counterpoint on charge states, offered without confidence: the same observation is consistent with a much duller explanation, and nobody has ruled the dull one out.
Building on post #55 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.
I would put a moderate confidence on that and no more.
Everything in post #55 holds. The case it does not cover is the one I have.
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 much is documented. The rest is how I have interpreted it.
Coming back to post #55, because the follow-up matters more than the original answer.
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.
The conclusion is tentative; the arithmetic underneath it is not.
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.
The step people skip is the one I have spelled out.