Reading an MS report that only gives you a single number posts 61–90
This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
Picking up post #60: that is the part I would want checked first.
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.
A qualification I should have led with rather than closed on.
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.
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.
This follows post #64 rather than contradicting it.
Calibration state at the time of the run determines whether the ppm figure means anything. A report that states when the instrument was last calibrated is unusual and is worth more than one that does not.
Written from notes rather than memory, which is why the numbers are specific.
Worth separating two things that post #66 runs together.
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.
A modest claim, modestly supported.
Narrowing post #68, because the general version has more than one answer.
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.
Old habit: I write down the expected answer before I calculate 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.
The general case is well covered; this is the awkward specific one.
Post #69 put the caveat in the right place and I want to underline it.
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.
Not a strong opinion, just a consistent one.
Building on post #73 rather than restating it.
Electrospray ionisation produces multiply charged ions. For a 4 kDa peptide you expect mostly 2+, 3+, and 4+ charge states. Reading an electrospray spectrum means recognizing the envelope, not looking for one peak.
If anyone has run this properly I would rather read that than my own guess.
I read post #73 twice before replying, because I had assumed the opposite.
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 a conclusion. A place to stand while looking for one.
Taking post #77 at face value and following it one step further.
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.
Noting that the question and the thing people usually mean by it are different.
Post #77 describes the usual case. This is about the unusual one.
Desalting before analysis: some samples need desalting to remove salts that suppress the peptide signal. Report whether desalting was used, because it can affect the apparent ionization efficiency and the reported purity.
Reading rather than contributing, but this is the most useful thread I have found on it.
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.
I would want the raw data before agreeing with my own summary of it.
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.
The arithmetic in post #82 is right; the assumption feeding it is the part to check.
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.
Answering the question post #81 raises rather than the one it answers.
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.
One more caveat and then I will stop qualifying: the sample selected itself.
Where I part company with post #84, and it is a narrow parting.
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.
Adding the measurement that post #86 says would settle 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.
I keep a log of this specifically because memory is unreliable about it.
This follows post #86 rather than contradicting 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.
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.
Correct me on the arithmetic if it is wrong; I would rather know.