MS/MS sequence confirmation: what it costs and when it is worth it — what changed since

Posting this under the heading it deserves: MS/MS sequence confirmation: what it costs and when it is worth it — what changed since Everything below is what sits behind that.

Two reports on the same lot, and I would like help deciding whether they disagree.

The first gives 98.6% by reversed-phase at 220 nm. The second gives 97.6% by a method described only as HPLC. Neither states the integration convention.

My instinct is that this gap is method rather than material. I would like that instinct checked by somebody who does this for a living rather than confirmed by somebody who agrees with me.

I read the opening post twice before replying, because I had assumed the opposite.

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.

Written in the hope of being told what I have missed.

Isotope pattern is a check that costs nothing to look at. A species at this molecular weight has a characteristic envelope, and something with the wrong envelope is not what it says it is.

Not the whole picture, but the part of it I can speak to.

Noted, and I have changed what I was going to do on the strength of it.

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.

Everything in post #2 holds. The case it does not cover is the one I have.

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.

That matches what I was told, which is not the same as knowing it.

Taking post #5 at face value and following it one step further.

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.

Adding the caveat now so it does not have to be extracted later.

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.

Noting that I have skin in this question and have tried to discount for it.

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.

A mass match establishes that the measured mass is consistent with the proposed composition. It does not establish purity, sequence order, or the absence of an isomer, and all three are frequently claimed from it.

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.

Marking that as an opinion rather than a finding.

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 put the burden of proof on the interesting explanation, not the dull one.

Where I part company with post #9, and it is a narrow parting.

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.

Tandem mass spectrometry: MS/MS fragments the molecular ion and uses fragment masses to confirm identity and detect modifications. A simple identity confirmation by LC-MS does not address post-translational modifications or impurities with the same or very close mass.