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.
Distinguishing a deletion sequence by mass alone Writing it up because I had to work it out twice and would rather nobody else did.
Trying to establish the boundary conditions on distinguishing a deletion sequence. The general claim is fine; I want to know where it stops holding, because that is where I am operating.
Three edge cases below, in increasing order of how far outside the usual range they sit.
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.
Adding a source would improve this post and I do not have one to hand.
Adding the measurement that the opening post says would settle it.
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.
Not disagreeing with anyone above, just adding the bit I keep having to look up.
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.
On post #5 — agreed on the reasoning, with one qualification.
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.
It is one reading of the data and not the only reasonable one.
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).
Posting it because the silence on this was starting to look like agreement.
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.
Second-hand, so weight it accordingly.
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.
Adding it because I spent an afternoon working it out and nobody should have to twice.
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.
For what it is worth, the same held on the two occasions I checked.
Confirming post #10 from a second method, which matters more than confirming it from a second person.
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.
Adding the measurement that post #13 says would settle 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.
A methods point on distinguishing a deletion sequence rather than a substantive one: if the comparison is not like for like, the difference you are measuring is the difference in method.
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.
Adding it in case it saves somebody the afternoon it cost me.
Where I part company with post #16, and it is a narrow parting.
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.
Taking post #18 at face value and following it one step further.
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.
The general case is well covered; this is the awkward specific one.
Post #16 and I disagree about the size of the effect, not about the direction.
Taking distinguishing a deletion sequence seriously for a moment rather than deflecting: the honest position is that the community has observations and no controlled comparison, and those two things support very different sentences.
Post #17 put the caveat in the right place and I want to underline it.
Distinguishing a deletion sequence is a good example of a question where the honest answer is boring and the interesting answers are unsupported. I would go with boring.
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.
Genuinely open to being wrong about this one.
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 number is defensible. The precision I gave it is not.
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.
Filing this under things that are true until someone shows me otherwise.
Nothing to add on the substance. Thank you for taking the question at face value.
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 #25 is right; the assumption feeding it is the part to check.
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.
Trifluoroacetate adducts are common in material purified with TFA and are one reason a mass spectrum from a peptide can look busier than expected.
One of those cases where knowing the mechanism does not help the decision.
Confirming post #28 from a second method, which matters more than confirming it from a second person.
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.