Charge states for a 4 kDa peptide, worked through posts 121–147
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.
The thing about charge states that took me longest to accept is that a plausible mechanism is not evidence of an effect. It is a reason to look, not a result.
I read post #121 twice before replying, because I had assumed the opposite.
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 claim is narrower than it sounds, and deliberately so.
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.
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.
That has held every time I have looked, which is not the same as always.
Building on post #125 rather than restating it.
Charge states: I have looked for the primary source twice and failed twice. Either it does not exist or it is somewhere I do not know to look, and I would like to know which.
Two questions I would want answered before drawing anything from the charge states data above: how were the cases selected, and what happened to the ones that dropped out.
On post #125 — agreed on the reasoning, with one qualification.
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.
It cost nothing to check and would have cost something not to.
Picking up post #129: that is the part I would want checked first.
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.
Two sources, same conclusion, and I could not rule out that one copied the other.
The useful distinction on charge states is between what was measured and what was inferred from it. Both end up in the same sentence and only one of them has error bars.
On post #130 — agreed on the reasoning, with one qualification.
Speaking only to charge states as I have actually seen it, rather than as it is usually described: the effect is real, it is smaller than the thread suggests, and the variance between people is larger than the effect.
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.
Take the reasoning and check the arithmetic; I do not always get it right.
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.
The honest answer is that it depends, and here is what it depends on.
Right, and stated more narrowly than I would have dared to state 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.
Happy to be the one who is wrong here if it settles the question.
The arithmetic in post #136 is right; the assumption feeding it is the part to check.
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.
That is the version I use. It may not be the version that is correct.
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.
I am not the right person to answer the follow-up to this.
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.
Collapsed as off-topic by two members at trust level 3 or above
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.
That distinction has done more work for me than anything else in this category.
I had written a reply contradicting post #139 and deleted it. Here is what survived.
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 that is already documented somewhere, ignore me and link it.
Noted, and thank you for writing it out rather than summarising it.
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.
That is all I can say without guessing.
The arithmetic in post #143 is right; the assumption feeding it is the part to check.
Adding a reference point for charge states. Mine is a single case, collected without controls, and I am posting the method alongside it so it can be discounted appropriately.
Where I part company with post #143, and it is a narrow parting.
I would call the community position on charge states likely rather than established, and I would be comfortable defending that hedge.
This topic was referenced in
- Distinguishing a deletion sequence by mass alone — the long versionAnalytics › Mass spectrometry · 44 replies
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