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.
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 answer changed when I changed how I was measuring, which was informative.
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.
Worth reading the earlier posts in this thread before acting on mine.
Post #30 put the caveat in the right place and I want to underline it.
What I would want before treating High-resolution as settled: the method, the sample, and whether anyone tried to find the opposite result. Two of the three are usually missing.
The arithmetic in post #33 is right; the assumption feeding it is the part to check.
Marking my uncertainty on High-resolution 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.
Mass error in parts per million is (observed minus theoretical) divided by theoretical, times a million. On a high-resolution instrument a low single-digit figure is unremarkable and expected.
I would put the burden of proof on the interesting explanation, not the dull 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.
Marking that as an opinion rather than a finding.
Staff rationale: Hidden by community flags. The claim about a named supplier was not accompanied by a batch, a date, a method or a document, which R6 requires.
Trifluoroacetate adducts are common in material purified with TFA and are one reason a mass spectrum from a peptide can look busier than expected.
The corresponding entry is in the public moderation log. Hidden posts are never deleted.
Post #38 describes the usual case. This is about the unusual one.
The version of High-resolution that circulates here is a simplification of a simplification. It is not wrong, but it has lost the conditions under which it holds, and those conditions are where the interesting cases live.
Post #40 answers the question as asked. The question underneath it is different.
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.
If that reads as pedantic, it is, and it has saved me twice.
Two people in this thread mean different things by High-resolution and are disagreeing about the definition while believing they are disagreeing about the facts. Worth pausing to define 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.
Coming back to post #43, because the follow-up matters more than the original answer.
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.
This is the version I would want a new member to read first.
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 has been true for the cases I have seen and I have not seen many.
Genuine question rather than a rhetorical one: has anyone here actually observed High-resolution, as opposed to read about it? The thread is long and I cannot tell.
Adding thanks rather than a view. I do not have a view worth the space.
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 literature is thinner on this than the confidence in the thread implies.
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.
If this contradicts something upthread, the upthread version may well be the better one.
This follows post #50 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.
Not the answer, but possibly the question that gets there.
Worth separating two things that post #52 runs together.
The reason High-resolution 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.
Taking post #54 at face value and following it one step further.
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.
Somebody will have a better source than mine, and I hope they post it.
Mass error in parts per million is (observed minus theoretical) divided by theoretical, times a million. On a high-resolution instrument a low single-digit figure is unremarkable and expected.
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.
On reflection I would soften that slightly.
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