On the subject in the title: Distinguishing a deletion sequence by mass alone — the long version Working notes rather than a conclusion.
A narrow question about Distinguishing a deletion sequence, 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.
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.
That holds for the case as described. Change the assumptions and it may not.
Building on post #2 rather than restating it.
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).
The opening post put the caveat in the right place and I want to underline it.
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.
I am not the right person to answer the follow-up to this.
Post #2 answers the question as asked. The question underneath it is different.
Two sentences on Distinguishing a deletion sequence and then I will stop, because the rest is speculation and the thread is better without mine.
What is documented is narrow. What is inferred from it is broad. The gap between them is where every argument here lives.
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.
The honest answer is that it depends, and here is what it depends on.
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.
Post #4 and I disagree about the size of the effect, not about the direction.
Adducts — sodium and potassium especially — shift the observed mass in predictable increments. A report showing an unexplained plus twenty-two is usually showing you a sodium adduct.
Post #6 is right about the mechanism and I think understates the practical bit.
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.
Coming back to post #8, 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.
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.
Anyone who has looked at this more carefully, please correct the record.
Worth separating two things that post #11 runs together.
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.
Take it as a starting point and not as a specification.
On post #15 — agreed on the reasoning, with one qualification.
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.
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.
The mechanism is plausible, which is not the same as established.
Noted, and I have changed what I was going to do on the strength of it.
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.
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.
The arithmetic in post #20 is right; the assumption feeding it is the part to check.
An acylated peptide has a mass that reflects the modification, so comparing against the mass of the unmodified backbone gives a mismatch that is not an error.
Two sources, same conclusion, and I could not rule out that one copied the other.
Answering the question post #22 raises rather than the one it answers.
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.
Confirming post #24 from a second method, which matters more than confirming it from a second person.
Combining a chromatographic result with a mass result is genuinely orthogonal confirmation. Either alone leaves a specific class of problem invisible, and the two classes do not overlap much.
Adding it in case it saves somebody the afternoon it cost me.
Helpful, and short, which on this subject is harder than long.
Post #27 describes the usual case. This is about the unusual one.
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.
Happy to be corrected if someone holds better data than mine.
Taking post #28 at face value and following it one step further.
An acylated peptide has a mass that reflects the modification, so comparing against the mass of the unmodified backbone gives a mismatch that is not an error.
If the premise is wrong, everything after it is decoration.
Post #27 and I disagree about the size of the effect, not about the direction.
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.
The claim is narrower than it sounds, and deliberately so.