Crosslinking Quantification

Crosslinking Quantification swatibanerjee060  2021-06-04
I am using Skyline for quantifying the cross-link and in many cases the precursor mass is not matching with raw file's precursor mass, though I am putting right modification. What could be the reason?
Nick Shulman responded:  2021-06-04
Can you send us your Skyline document?

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file is less than 50MB you can attach it to this support request. Otherwise, you can upload it here:

I am not sure I understand your question, but it sounds like I might also need to look at your raw file so please send that to if you think the raw file might be relevant.

-- Nick
swatibanerjee060 responded:  2021-06-15
I have uploaded one screenshot. For this cross-link the precursor mass is showing 487.0324 (+4) but in Proteome Discoverer and also in Merox I am getting precursor mass as 487.0391. What could be the reason?
Brendan MacLean responded:  2021-06-15
We would need your Skyline document using File > Share to save a

It is impossible to know even the chemical formula of your crosslinker from just the screenshot.

swatibanerjee060 responded:  2021-06-15
I have uploaded the zip file here.
Brendan MacLean responded:  2021-06-15
Can you also validate that Skyline and PD give matching m/z values for precursors of the separate unlinked peptides in the example you chose?

This would give us clearer evidence that it is something in the linking and not just that Skyline and PD disagree on basic atomic masses.

Thank for your help in isolating this issue.

Brendan MacLean responded:  2021-06-15
Something I find strange about your linker definition is that it has the following formula:


And yet claims to have the potential for Deuterium loss. Does the linker contain deuterium? If so, then its chemical formula is not correct. It can't both have the above chemical formula and allow Deuterium loss.

If the true chemical formula contains Deuterium but not in Skyline, that could lead to a slight difference in m/z like the one you have noticed.
swatibanerjee060 responded:  2021-06-16
Thank you for your reply. Yes, it was my mistake to put Deuterium loss in the settings. The cross-linker doesn't have any Deuterium. I have made correction . Still I am getting the same result, slight different precursor mass . I have also checked the precursors of unlinked peptides in both PD and Skyline. Both are giving same mass. So, its something in linking for which I am getting slight different mass after cross-linking.
Nick Shulman responded:  2021-06-16
The difference in m/z between 487.0324 and 487.0391 is .0067. Since those precursor ions are charge 4, that means we are probably looking for a mass difference of 0.0268.

I cannot think of anything that would cause that sort of mass difference.

If you're saying that the mass difference only shows up with your crosslinked peptides, then it must mean that there is a difference between what Skyline thinks the chemical formula and mass of your crosslinker is, compared to what Proteome Discoverer thinks it is.

I do not know how to use Proteome Discoverer, but if you send us your Proteome Discoverer files (maybe .msf or .pdresult?) I might be able to figure out what is going on.
-- Nick
Brendan MacLean responded:  2021-06-16
Just to check Nick's assertion on the mass delta, are you able to get PD's m/z for other charge states, e.g. 1, 2, 3, as you show for Skyline in your original screenshot? As Nick suggests, we would expect to see a mass difference of 0.0268 / charge or:

1 - 0.0268
2 - 0.0134
3 - 0.0089
4 - 0.0067
swatibanerjee060 responded:  2021-06-17
Hello Nick & Brendan,

In PD I got 2 charge states (+3 & +4) for this linkage.
For charge state 4, m/z = 487.0391 (PD), 487.0324 (Skyline) ; mass difference 0.0268
For charge state 3, m/z = 649.0482 (PD), 649.0407 (Skyline) ; mass difference 0.0225
I have attached one PD result file here.

Nick Shulman responded:  2021-06-17

Can you try sending us that .msf file again?
I can see that you did upload the file "201204_BSA_BS3_100_SET_1.msf", but, for some reason, I cannot actually find that file in the place that it was supposed to have been uploaded.
I will also send you an email message directly in case it's easier to send files over email.
-- Nick
swatibanerjee060 responded:  2021-06-17
Hi Nick,

I have uploaded it again. If you are not still able to find it, I will send it over email.

Brendan MacLean responded:  2021-06-17
It would be great to get a few more comparable m/z values for the same cross-linked peptides and multiple charge states from PD.

The expected formula for calculating the neutral mass from a m/z is:

neutral mass = mz * z - proton mass * z

Skyline uses a proton mass of 1.007276 in calculating m/z from a neutral mass. If you apply the formula above to the Skyline values you get neutral mass values within 0.00023 (rounding error at this precision). In order to get identical neutral masses from the two m/z values you have supplied for PD, I need to use a proton mass of 1.0118. No idea where this value might come from, but I would be interested to see if it holds across more precursor m/z values for different charge states of the same molecule.
Nick Shulman responded:  2021-06-17


I think the number 487.0391 that you are seeing in ProteomeDiscoverer might just be the m/z that the mass spectrometer measured, and not the theoretical m/z calculated from the chemical formula.

Here is some of the data that I see in the table "HlxlMs2Scanss":


The difference between the numbers in the Theoretical Mass column and the PrecursorMonoisotopicMass column is very close to 0.0268. It appears that the values in the TheoreticalMass column agree with what Skyline thinks the neutral mass of that crosslinked peptide is, except that the numbers in that table seem to have had the mass of a proton added to them.

As far as I can tell, Skyline and ProteomeDiscoverer agree as to what the mass of that crosslinked peptide is, but the numbers you are looking at are the numbers that were measured by the mass spectrometer, and which can always be expected to be off by a bit.

-- Nick