What I would want before treating Charge states as settled: the method, the sample, and whether anyone tried to find the opposite result. Two of the three are usually missing.
2 likes 4mo
Analytics · Mass spectrometry · continued
[2026 update] Charge states for a 4 kDa peptide, worked through posts 61–90
This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
Answering the question post #58 raises rather than the one it answers.
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.
This has been discussed before and I could not find the thread, so, again.
8 likes 4mo
n.villalobos, post #23: Building on post #20 rather than restating 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. Go to post
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.
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.
0 likes 4mo
b.jansen, post #52: 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. Go to post
Taking Charge states seriously for a moment rather than deflecting: the honest position is that the community has observations and no controlled comparison, and those two things support very different sentences.
ppm_error, post #17: I read post #16 twice before replying, because I had assumed the opposite. 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. That holds for the case as described. Change the… Go to post
Post #64 is the version of this I will quote in future. One addition.
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.
I have left out the parts I could not verify.
Birkeland, post #32: 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. Go to post
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.
h.bhattacharya, post #64: 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. Go to post
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.
g.valckenaere, post #8: 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. Go to post
Answering the question post #70 raises rather than the one it answers.
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 this contradicts something upthread, the upthread version may well be the better one.
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.
18 likes 4mo
Post #73 is the version of this I will quote in future. One addition.
I think the Charge states question is answerable and has not been answered, which is a more optimistic position than most of this thread.
7 likes 4mo
Everything in post #73 holds. The case it does not cover is the one I have.
Charge states sits at the boundary between what this community can usefully discuss and what it cannot, and I think it falls on the discussable side, narrowly.
1 like 4mo
h.nicolaides, post #59: Noted, and I have changed what I was going to do on the strength of it. Go to post
Narrowing post #73, because the general version has more than one answer.
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.
The strength of my opinion here exceeds the strength of my evidence.
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.
0 likes 3mo
I would rather this thread reach "we do not know" about Charge states than reach a confident answer that nobody can support when asked.
19 likes 3mo
Trifluoroacetate adducts are common in material purified with TFA and are one reason a mass spectrum from a peptide can look busier than expected.
Someone should write this up properly, and it should probably not be me.
1 like 3mo
vial_desk, post #49: Seconded. It reads as careful rather than confident, which is the right register. Go to post
Picking up post #82: that is the part I would want checked first.
Reading back through the Charge states threads from last year, the same three questions come up every time and only one of them has ever been answered properly. That seems like a documentation gap rather than a knowledge gap.
On post #80 — agreed on the reasoning, with one qualification.
Charge states is well covered in the tag pages, and the older discussions are better than the recent ones because they were argued out properly. Worth twenty minutes before adding to this one.
25 likes 3mo
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 want the raw data before agreeing with my own summary of it.
0 likes 3mo
Coming back to post #84, because the follow-up matters more than the original answer.
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.
0 likes 3mo
Collapsed as off-topic by two members at trust level 3 or above
This follows post #86 rather than contradicting it.
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 evidence for this is thinner than the way I have phrased it suggests.
bias_variance, post #80: I would rather this thread reach "we do not know" about Charge states than reach a confident answer that nobody can support when asked. Go to post
If someone has run Charge states properly I would rather read that than my own reconstruction of it. Posting mine only because the thread has gone quiet.
Post #86 answers the question as asked. The question underneath it is different.
Where I have landed on Charge states, having got it wrong once in public: the direction is clear, the magnitude is not, and anyone quoting a precise magnitude has borrowed it from somewhere that did not measure it.
1 like 3mo
I read post #88 twice before replying, because I had assumed the opposite.
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.
Noting that I have skin in this question and have tried to discount for it.
7 likes 3mo