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.
Caveat: everything above assumes the paperwork is what it says it is.
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Analytics · Mass spectrometry · continued
Adducts: sodium, potassium, and the peak you did not expect posts 61–78
This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
cohort_watch, post #23: If you are new and reading this thread for the answer to adducts: the answer is conditional, the conditions are in the third reply, and the rest of the thread is worth skipping. Go to post
Before the thread moves on from adducts — what is the sample size behind the claim? I am not being difficult; I have seen the same figure quoted from an n of four and from an n of four hundred.
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.
The disagreement above is smaller than it looks once the terms are fixed.
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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.
The number is defensible. The precision I gave it is not.
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b.jankowiak, post #31: My experience of adducts contradicts the reply above. I am posting it as a data point rather than as a refutation, because one person's experience is exactly that. Go to post
Adducts is one of those subjects where the general answer and the answer for a specific case diverge, and the thread will go in circles until someone says which one is being asked for.
Collapsed as off-topic by two members at trust level 3 or above
On post #65 — agreed on the reasoning, with one qualification.
Two things can be true about adducts at once: the mechanism is plausible and the evidence for the size of the effect is thin. Most of the argument here is people defending the first against attacks on the second.
Picking up post #65: that is the part I would want checked first.
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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On adducts, I would rather understate and be corrected upward than overstate and be quoted. That is a house style here and it is a good one.
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On adducts I would separate what is worth knowing from what is worth acting on. The first list is long and the second is short, and conflating them is how threads get heated.
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Post #70 put the caveat in the right place and I want to underline 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.
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b.nwosu, post #20: Having read the whole adducts thread before replying: the question in the first post has not actually been answered yet, and three of us have answered a nearby one instead. Go to post
The arithmetic in post #72 is right; the assumption feeding it is the part to check.
The most useful reply I ever got about adducts was a request to state my units. It sounds like pedantry and it has saved me 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.
Posting it because the silence on this was starting to look like agreement.
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l.aaltonen, post #13: 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 is where I would start, not where I would stop. Go to post
Adding the measurement that post #76 says would settle it.
Trifluoroacetate adducts are common in material purified with TFA and are one reason a mass spectrum from a peptide can look busier than expected.
I have left out the parts I could not verify.
b.jankowiak, post #31: My experience of adducts contradicts the reply above. I am posting it as a data point rather than as a refutation, because one person's experience is exactly that. Go to post
Post #74 describes the usual case. This is about the unusual one.
The honest answer on adducts is that it depends, and the useful part is the list of what it depends on. Four items, in rough order of how much they matter.
Most people get the first two right and then argue about the fourth.
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- Isotope patterns and why the monoisotopic peak is not the tallest — does this still hold?Analytics › Mass spectrometry · 2 replies
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