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.
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Analytics · Mass spectrometry · continued
[2026 update] Charge states for a 4 kDa peptide, worked through posts 61–90
This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
On post #58 — 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.
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n.villalobos, post #23: This follows post #20 rather than contradicting it. 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. Go to post
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.
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b.jansen, post #52: post #51 is right about the mechanism and I think understates the practical bit. 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. Go to post
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.
ppm_error, post #17: I read post #15 twice before replying, because I had assumed the opposite. 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. Go to post
This follows post #64 rather than contradicting it.
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.
Birkeland, post #32: 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. Go to post
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.
Collapsed as off-topic by two members at trust level 3 or above
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.
h.bhattacharya, post #64: 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. Go to post
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.
g.valckenaere, post #8: 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. Go to post
I read post #69 twice before replying, because I had assumed the opposite.
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.
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.
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post #73 is right about the mechanism and I think understates the practical bit.
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.
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Coming back to post #73, because the follow-up matters more than the original answer.
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.
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h.nicolaides, post #59: 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. Go to post
Picking up post #73: that is the part I would want checked first.
For anyone arriving from a search: the marked solution above is the direct answer, and the replies underneath it add the caveats that make it safe to use.
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.
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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.
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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.
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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.
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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.
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vial_desk, post #49: For anyone arriving from a search: the marked solution above is the direct answer, and the replies underneath it add the caveats that make it safe to use. Go to post
post #82 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.
On post #80 — 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.
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I read post #84 twice before replying, because I had assumed the opposite.
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.
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Collapsed as off-topic by two members at trust level 3 or above
post #86 is right about the mechanism and I think understates the practical bit.
I disagree with the reply above, and I think the disagreement is substantive rather than terminological.
The distinction being drawn does not survive when you look at the published data for this specific question. I would be glad to be shown wrong on this, because the version I am arguing against is more convenient.
bias_variance, post #80: 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. Go to post
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).
Picking up post #86: that is the part I would want checked first.
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.
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Coming back to post #88, 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.
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