Theoretical peptide z-ion mass appears to be incorrect

Theoretical peptide z-ion mass appears to be incorrect JHamey  2018-11-14

Hi there Skyline team,

I've come across a problem to do with the theoretical mass of z-ions in Skyline. They appear to be consistently off by 1 Da - they are predicted to be 1 Da lighter than they actually are. E.g. a z13 ion is predicted to be 1316.7 Da when it is actually 1317.7 Da. When I searched for threads related to this issue I came across this one (Issue 498):

It appears as if the "fix" that was made to make z-ions 1 Da lighter was done in error. The 1 Da heavier masses for z-ions I am getting from MS-Product match what I see experimentally for a normal peptide (it was noted in that thread that the peptide where the "error" was observed contained an abnormal gamma linkage at the position where the mass shift was thought to be wrong). C-ion masses are perfectly ok. I see this consistently for at least 5 different z-ions in the peptide.

Thank you in advance for your help.

Nick Shulman responded:  2018-11-14
The numbers calculated in Skyline match what's on this web site:
They also match what's in the supplemental data for this paper:

Do you have another tool which calculates different numbers for Z-ions? Can you send us a screenshot of that?

Skyline subtracts an NH3 from a y-ion in order to get the mass of the z-ion. When I look at the molecule diagram on the Wikipedia page, I can't figure out why that would be the correct thing to do, but it does match all of the results that I can find online.

If you'd like, you can send us your Skyline document. There might be something else going on.
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files, including extracted chromatograms.

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

You should also send us your raw file so we can see those MS2 spectra.
-- Nick
JHamey responded:  2018-11-14
Thanks for the quick response Nick.

I got the masses for z-ions from MS-Product ( See the attached image for the spectrum with matching z-ions.

However, I just went and searched my data with Mascot and it appears what MS-Product calls z-ions are in fact z+1 ions (see attached image). Matrix science even says that ETD generally produces z+1 ions ( and so Mascot didn't even search for z-ions. So it appears as if the mass calculation for z-ions is correct, but the problem is that what ETD produces (at least for lower charge state peptides) is mostly z+1 ions. So is it possible to use z+1 ions instead of z-ions?

Thank you.
Brendan MacLean responded:  2018-11-18
The Mascot image you sent appears to have z+2 also. It looks like MS-Product allows c+2, c+1, c, c-1 and z, z+1, z+2, z+3. So, maybe the right solution is to also add these to the list of ions that Skyline supports. If so, probably we should also add a pop-up checklist attached to a menu button like we have for chemical formulae. Do you have any more information on the need all 8 c- and z-ion options? Or should we contact the authors of Mascot and MS-Product for a better sense of the complete range we should support for c- and z-ions?

Thanks for pointing this out to us.

Brendan MacLean responded:  2018-11-18
I read the page from Matrix Science where they say, "Electron Transfer Dissociation and Electron Capture Dissociation generate primarily c, y, z+1, and z+2 ions. In some cases, w ions can also be prominent." So, maybe ETD users only need z+1 and z+2 added to Skyline? And we can wait for some other use case to require the other 4 c and z ions (c+2, c+2, c-1, z_3).

Does that seem like it would be enough for you to achieve what you were hoping for in your Skyline use for this experiment?

JHamey responded:  2018-11-18
That would be fantastic - certainly at least having z+1 and z+2 would be really good. Certainly I do see at least c+1 and c+2 ions, but since there's always the c ion as well it's not super important from what I've seen, whereas since it appears that the z+1 ion appears without the corresponding z ion, having that added would be really useful.

Thanks for the help with this.
Brendan MacLean responded:  2018-11-20
Okay. I think I understand a bit better after running this by David Creasy at Matrix Science (Mascot developer). There is a difference in nomenclature between Matrix Science (Biemann) and Protein Prospector (UCSF) which is explained by UCSF here:

With some further elaboration of how these ions show up in ETD experiments here:

Where the paper refers to Biemann z+1 and z+2 (and c and c-1) in their tables when they use z• and z+1. So, in Matrix Science/Biemann nomenclature (which Skyline seems to have adopted on March 1, 2017 after a prior support request to change z-ion calculation from -NH2 to -NH3), when you have z+2, you might also have c-1, but the probability of c-1 appears to be very close to noise (measured by x) in the tables of the paper.

I am not sure you are really seeing c+1 and c+2 as ions per se since the +1 and +2 are supposed to refer to hydrogen. The c+1 and c+2 you see, if you always see them with c, may simply be the heavy isotopes of the c-ions caused mostly by 13C in the molecule, and Skyline never targets those for any other ion type.

I think we will stay with Matrix Science/Biemann nomenclature to avoid flip-flopping on this, and we will try to soon add support for z+1 and z+2, which appear to be true monoisotopic ion forms that appear in ETD, though z+2, according to Devid Creasy, will only be seen with charge 3+ and above. Though, I am now curious what the nature of the experiment was that caused the prior support request when our z-ion calculation was actually calculating z+1, and Biemann z ions don't seem to appear in ETD and ECD.
JHamey responded:  2018-11-20
That all makes a lot of sense. I think you're completely right that the c+1, c+2 ions are just isotopic peaks. Their distribution looks like what I see in HCD/CID data too.

I think the previous request was unusual as it wasn't a normal peptide bond being analysed - so maybe this affected the likelihood of seeing z+1 ions instead of z• ions with ETD fragmentation at that bond.

Thanks again for looking into this.