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 · go to the accepted answer.
Adding what did not work for me on charge states, since the failures never get written up and they are half the useful information.
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.
On post #91 — agreed on the reasoning, with one qualification.
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.
I have deliberately not rounded that, because the rounding is where the argument starts.
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Picking up post #95: that is the part I would want checked first.
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.
The reason charge states is hard to answer is that the obvious measurement and the relevant quantity are not the same thing, and substituting one for the other is silent.
The bit of charge states that nobody enjoys is that the answer changes depending on what you are trying to decide with it. Say what the decision is and the thread will converge.
This follows post #99 rather than contradicting it.
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 been on both sides of the charge states argument in this category within eighteen months, which should tell you how strong the evidence for either side is.
That is clearer than the version I had in my head. Thank you.
Adding a small correction to the charge states summary above rather than a disagreement with it. The substance holds; one of the figures is out by a factor that matters.
The arithmetic in post #103 is right; the assumption feeding it is the part to check.
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.
Where I part company with post #103, and it is a narrow parting.
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.
Second-hand, so weight it accordingly.
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.
Scoping that to what I have actually seen rather than what I have read.
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.
It reads as pedantry until the day it does not.
Posting my charge states numbers with the method attached so they can be discounted properly. Uncontrolled, unblinded, and collected by someone who wanted a particular answer.
Filing a mild objection to the consensus on charge states. Mild because I might be wrong; an objection because nobody has addressed the case that does not fit.
Confirming post #107 from a second method, which matters more than confirming it from a second person.
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 have left out the parts I could not verify.
What would change my mind on charge states is a second dataset collected by someone with no stake in the first. Until then I hold it loosely and I would rather say so than pretend to more.
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Building on post #110 rather than restating it.
Two claims get bundled together under charge states and they need separating. The descriptive one — this is what was observed — is usually well supported. The causal one — this is why — usually is not.
Almost every disagreement in threads like this one dissolves once you say which of the two you are making.
Post #112 put the caveat in the right place and I want to underline 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.
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 would rather say I do not know than round it up to an answer.
Everything in post #112 holds. The case it does not cover is the one I have.
On charge states, the part that usually goes wrong is that the question is asked as though it has one answer. It has a range, and the width of the range is the interesting bit.
If you can post the two or three numbers you are working from, several people here will check the arithmetic rather than argue about the conclusion.
Taking post #114 at face value and following it one step further.
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.
Post #118 answers the question as asked. The question underneath it is different.
For anyone finding this later: the short answer on charge states is that it depends on one thing, and the rest of the thread is people identifying which thing.
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.