Distinguishing a deletion sequence by mass alone — the long version posts 31–45

Where I part company with post #29, and it is a narrow parting.

Sample preparation for mass spectrometry can itself introduce modifications, particularly oxidation. A finding at trace level may be telling you about the preparation rather than the material.

Seconded. It reads as careful rather than confident, which is the right register.

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.

It is the sort of thing that seems obvious in retrospect and was not at the time.

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.

The evidence for this is thinner than the way I have phrased it suggests.

Building on post #33 rather than restating it.

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.

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.

A modest claim, modestly supported.

Common adducts: sodium adds ≈22, potassium adds ≈38 compared to hydrogen. A [M+Na]+ peak is common and its mass is predictable from the base mass.

Written from notes rather than memory, which is why the numbers are specific.

Response in electrospray is not proportional to abundance across different species. Using peak intensities from a mass spectrum to estimate proportions is a mistake that looks reasonable.

The interesting part of this is the exception, and I do not understand the exception.

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.

Worth one more sentence than it usually gets.

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.

The general case is well covered; this is the awkward specific one.

Picking up post #42: that is the part I would want checked first.

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.

If anyone has run this properly I would rather read that than my own guess.

On post #40 — agreed on the reasoning, with one qualification.

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.

Not a strong opinion, just a consistent one.