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 · go to the accepted answer.
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.
The general answer and the answer for your case may diverge here.
An honest declaration on charge states: I have a prior here and it is strong enough that you should weight what I say downward. Stating it rather than hiding it.
Coming back to post #62, because the follow-up matters more than the original answer.
Charge states is worth one more sentence than it usually gets, and the sentence is the one about how the number was arrived at.
Taking post #64 at face value and following it one step further.
The arithmetic for a doubly charged species is (M + 2 x 1.00728) / 2, and the analogous expression for higher charge states. Working it through once makes the reported values legible.
The uncertainty is in the assumption, not in the calculation.
Post #62 and I disagree about the size of the effect, not about the direction.
What I would tell a new member reading about charge states for the first time: the confident posts are not the reliable ones, and the reliable ones are longer.
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.
I think the charge states question is answerable and has not been answered, which is a more optimistic position than most of this thread.
Post #69 put the caveat in the right place and I want to underline it.
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.
The evidence for this is thinner than the way I have phrased it suggests.
Building on post #69 rather than restating 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.
On reflection I would soften that slightly.
I would put moderate confidence on the mainstream reading of charge states and no more. That is not scepticism for its own sake; it is where the sourcing actually stops.
Taking post #73 at face value and following it one step further.
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.
The interesting part of this is the exception, and I do not understand the exception.
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.
Worth one more sentence than it usually gets.
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.
Reading back through the charge states threads from last year, the same three questions come up every time and only one of them has ever been answered properly. That seems like a documentation gap rather than a knowledge gap.
Narrowing post #78, because the general version has more than one answer.
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.
That holds under the stated conditions and I have stated them.
Everything in post #80 holds. The case it does not cover is the one I have.
Distinguishing three things in the charge states discussion that keep getting used interchangeably: the observation, the proposed mechanism, and the recommendation that gets attached to both.
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 general case is well covered; this is the awkward specific one.
Trifluoroacetate adducts are common in material purified with TFA and are one reason a mass spectrum from a peptide can look busier than expected.
The strongest argument against my own position on charge states, stated as well as I can state it, since nobody else has yet.
Collapsed as off-topic by two members at trust level 3 or above
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.
The practical version of charge states is three sentences long. The rigorous version is three pages and reaches the same conclusion with the conditions attached.
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.