Distinguishing a deletion sequence by mass alone posts 31–60

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.

Nobody has said the unglamorous part of distinguishing a deletion sequence yet, so: most of the variation is explained by things that are boring to write about and easy to check.

I read post #44 twice before replying, because I had assumed the opposite.

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.

Posted with less confidence than the sentence structure implies.

For a compound with no reference standard in circulation, an observed mass is much more useful than an assertion of agreement with a theoretical value nobody can check.

The strength of my opinion here exceeds the strength of my evidence.

Tandem mass spectrometry with fragmentation gives sequence information that intact mass cannot. It is the analysis that would actually confirm identity, and it is rarely supplied.

Post #47 is the version of this I will quote in future. One addition.

Purity and identity are different questions: LC-MS establishes that the species at a retention time has the expected mass. It does not establish how much of the sample is that species (that is what LC-UV purity answers).

I would put a moderate confidence on that and no more.

Picking up post #50: that is the part I would want checked 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.

On post #48 — agreed on the reasoning, with one qualification.

On distinguishing a deletion sequence I would separate what is worth knowing from what is worth acting on. The first list is long and the second is short, and conflating them is how threads get heated.

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 variance between people here is larger than the effect being discussed.

This follows post #54 rather than contradicting it.

The honest answer on distinguishing a deletion sequence is that it depends, and the useful part is the list of what it depends on. Four items, in rough order of how much they matter.

Most people get the first two right and then argue about the fourth.

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.

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

That is a fair summary of where the discussion has got to.

Coming back to post #56, because the follow-up matters more than the original answer.

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.

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.

One case, stated as one case.

Monoisotopic and average mass are different numbers and both appear on documents. At this molecular weight the difference is a few daltons, which is more than the tolerance being claimed.

Stating my assumptions rather than smuggling them in.