This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
Worth separating two things that post #27 runs together.
Sample matrix effects: if a sample is dissolved in a complex matrix, other compounds in the matrix can suppress the peptide signal. Clean samples give higher sensitivity than dirty samples.
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.
On post #31 — agreed on the reasoning, with one qualification.
Resolution: "high resolution" commonly means <5 ppm across the mass range. Unit-resolution instruments achieve ±1 Da at best and cannot distinguish two species differing by less than 1 Da in total mass.
post #35 answers the question as asked. The question underneath it is different.
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.
Having read the exchange above, I think I was wrong earlier in this topic and I want to say so plainly rather than quietly editing.
The correction was fair and I had been repeating something I had not checked carefully enough.
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.
Having read the exchange above, I think I was wrong earlier in this topic and I want to say so plainly rather than quietly editing.
The correction was fair and I had been repeating something I had not checked carefully enough.
post #39 is right about the mechanism and I think understates the practical bit.
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.
Practical note that does not fit anywhere else. Whatever you conclude from this topic, write down what you did and when. The single most useful thing in your own records is not any individual result; it is that they are dated and consecutive.
This follows post #40 rather than contradicting it.
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.
Thank you for the correction. I have edited my earlier post with a note rather than silently, so the thread still makes sense to read. The error was mine and it was the kind that comes from remembering a figure instead of looking it up.
Picking up post #44: that is the part I would want checked first.
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.
Coming back to post #46, because the follow-up matters more than the original answer.
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.
post #48 is right about the mechanism and I think understates the practical bit.
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.
Worth separating two things that post #46 runs together.
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.
On post #49 — agreed on the reasoning, with one qualification.
Charge states observed: for semaglutide (4113.6 Da) the doubly charged ion appears at m/z ≈ 2057, triply charged at ≈ 1371, quadruply at ≈ 1029. Those are the positions to look for; the heights depend on the ionization efficiency.
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.
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).
Worth separating two things that post #53 runs together.
Sample matrix effects: if a sample is dissolved in a complex matrix, other compounds in the matrix can suppress the peptide signal. Clean samples give higher sensitivity than dirty samples.
post #57 is right about the mechanism and I think understates the practical bit.
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.
Coming back to post #57, because the follow-up matters more than the original answer.
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.
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.