Calibration matters: a high-resolution instrument out of calibration can report mass with ppm error large enough to be uninformative. Check when the instrument was last calibrated before trusting the reported accuracy.
High-resolution MS on aggregates and what it can see — one year on Writing it up because I had to work it out twice and would rather nobody else did.
A narrow question about High-resolution, deliberately narrow, because the broad version has been asked here four times and produced four long threads and no answer.
One question, stated units, stated method, and what I have already ruled out.
Picking up the opening post: that is the part I would want checked first.
What mass accuracy establishes: the measured mass is consistent with a specific composition. What it does not establish: purity, sequence order, stereochemistry, or the absence of an isobaric species. Every one of those requires something else.
It is the sort of thing that seems obvious in retrospect and was not at the time.
Calibration matters: a high-resolution instrument out of calibration can report mass with ppm error large enough to be uninformative. Check when the instrument was last calibrated before trusting the reported accuracy.
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 have changed my mind on this once already, so take it as current rather than settled.
This follows post #5 rather than contradicting it.
High-resolution is a good example of a question where the honest answer is boring and the interesting answers are unsupported. I would go with boring.
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 holds under the stated conditions and I have stated them.
Post #5 and I disagree about the size of the effect, not about the direction.
Where I would push back on the High-resolution consensus is the confidence, not the direction. The direction looks right. The confidence is borrowed.
Small methodological point on High-resolution: repeating a measurement is cheap and resolves most of what is being argued about here at no cost to anyone.
Post #10 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.
I read post #9 twice before replying, because I had assumed the opposite.
Deamidation adds approximately one dalton and produces a species that frequently elutes very close to the parent. It is the hardest common impurity to see chromatographically and the easiest to see by mass.
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.
Two people can read the same figure differently here and both be reasonable.
The useful distinction on High-resolution 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.
High-resolution has been discussed here with more heat than it deserves, mostly because two definitions have been in play the whole time.
Right — I had this wrong and I am glad to have read it before it mattered.
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.
If anyone has run this properly I would rather read that than my own guess.
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 would rather be precise about what I do not know than vague about what I do.
I think the High-resolution question is answerable and has not been answered, which is a more optimistic position than most of this thread.
Post #17 describes the usual case. This is about the unusual one.
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.
Reading it back, the second half matters more than the first.
Adding the measurement that post #21 says would settle it.
What I would tell a new member reading about High-resolution for the first time: the confident posts are not the reliable ones, and the reliable ones are longer.
High-resolution 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.
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.
I would want to see it done twice before believing it once.
Deamidation adds approximately one dalton and produces a species that frequently elutes very close to the parent. It is the hardest common impurity to see chromatographically and the easiest to see by mass.
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.
Old habit: I write down the expected answer before I calculate it.