[2026 update] Charge states for a 4 kDa peptide, worked through posts 91–120
This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
The strongest argument against my own position on Charge states, stated as well as I can state it, since nobody else has yet.
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.
I would put the burden of proof on the interesting explanation, not the dull one.
Adding the measurement that post #91 says would settle it.
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.
I would rather post the uncertainty than round it away.
Coming back to post #91, because the follow-up matters more than the original answer.
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.
The answer changed when I changed how I was measuring, which was informative.
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.
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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.
Thank you for taking the time. That was more work than a reply usually is.
That matches what I have seen, for whatever a single anecdote is worth.
Post #96 answers the question as asked. The question underneath it is different.
Having read the whole Charge states thread before replying: the question in the first post has not actually been answered yet, and three of us have answered a nearby one instead.
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.
Picking up post #100: that is the part I would want checked first.
The failure mode on Charge states is boring rather than dramatic. It is almost always the step everyone assumes was done correctly because it is too simple to get wrong.
Coming back to post #100, because the follow-up matters more than the original answer.
Trifluoroacetate adducts are common in material purified with TFA and are one reason a mass spectrum from a peptide can look busier than expected.
One of those cases where knowing the mechanism does not help the decision.
What I can speak to on Charge states is narrow, so I will keep it narrow rather than generalising from it. Beyond that boundary I do not know.
Summarising the Charge states thread so far, since it is long and the answer is buried: the first reply has the method, the fourth has the correction to it, and the rest is people agreeing at length.
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I read post #103 twice before replying, because I had assumed the opposite.
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.
Post #106 answers the question as asked. The question underneath it is different.
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.
A qualification I should have led with rather than closed on.
Post #108 and I disagree about the size of the effect, not about the direction.
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.
If anyone has run this properly I would rather read that than my own guess.
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.
That has been true for the cases I have seen and I have not seen many.
Confirming post #110 from a second method, which matters more than confirming it from a second person.
My understanding of Charge states is a few years old and may have been superseded. If it has been, I would genuinely like to know rather than keep repeating it.
Charge states: I would want to see the raw numbers rather than the summary before agreeing. Summaries lose exactly the information that would settle this.
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.
The mechanism is plausible, which is not the same as established.
No disagreement from me. Posting only so the question does not look ignored.
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 am not the right person to answer the follow-up to this.
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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.
I would be interested in a counterexample if anyone has one.
Answering the question post #116 raises rather than the one it answers.
I have three months of notes on Charge states and the honest summary is that the trend is real and the week-to-week numbers are noise. I nearly drew the opposite conclusion from the first fortnight.
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.
Speaking for myself and not for anyone else who has posted here.
Post #116 put the caveat in the right place and I want to underline it.
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.
Anyone who has looked at this more carefully, please correct the record.