Points Across Peak

Points Across Peak Yasin  2018-11-11

Dear Skyline team,

when creating a report template it is possible to add the "points across peak" - column. However, this number only counts the number of data acquisition points. Often it would be nice to filter for peaks with a certain number of points above 0 in between the integration boarders as this would save the time of manually deleting peaks with less then a specific number of (connected) points above 0. Hope that is something you would consider to implement.

Anyway, thank you in advance!


Yasin responded:  2018-11-14

Did I explain well enough what I am talking about?

I will try to give an example to make it more clear. I also uploaded a table.

Let's say I extract metabolite A with two adducts with two isotopologues each. In sample 1 I see well shaped chromatographic peaks for each of those transitions. However, for sample two I only observe one peak at the M+H adduct at the highest isotopologue (M+0), while I only observe noise for the other transitions.
How could I possibly proceed now if I wanted the areas of those chromatographic peaks? Once I exportet a report I am not aware of any possibility to distinguish the area values which originated from those from those originating from chromatographic peaks.
Simply setting an area threshold is usually not sufficient.
I am also not aware of any other exportable variable that would help me with that. One way that would really help is to introduce a column that gives the number of consecutive scans > baseline and/or the number of consecutive scans > 0.

If I am just not seeing an already implemented solution to that problem I would be really happy if you could tell me. Taking notes outside of Skyline is the only (really really annoying and error prone) way I can think of at the moment. I think the problem I described here is one many people could run into at some point.

Thank you in advance,

Nick Shulman responded:  2018-11-14
Would the "Fwhm" value help? That's the Full Width at Half Max, which will tell you how wide the chromatogaphic peak is. That column is available on the Transition Result in the Document Grid. That would give you a good idea of whether the peak has only a single scan with high intensity.
There's another column, "Fwhm Degenerate" which tells you whether the apex of the peak is so close to one of the integration boundaries that there is no point below the half max of the peak.

I am not sure that looking at the number of points that are above the background would be a good measure of anything. Skyline defines the background as the intensity at the lower of either the start or end of the peak. For this reason, in completely random data, on average 2/3 points across the peak will be able the background.
-- Nick
Yasin responded:  2018-11-15
Thank you for the response.

I was using the FWHM column until now. The problem is that the scan range is not the same over a lc-ms when an orbitrap instrument is used. So in some cases there are relatively high and broad peaks with less than 5 points, while there are also peaks with about the same intensity and FWHM with about 10 points.

You are right that it would not make sense to check the number of points above the baseline to get rid of that problem. However, I believe that the max number of consecutive points above the baseline is very unlikely to occur randomly and would give a very clear picture in a data matrix.

Is that something that would make sense for you?

Yasin responded:  2018-11-17
I just discovered another issue using FWHM for that purpose:

When there are multiple spikes within the integration range Skyline is giving the distance between first and last spike as FWHM. I also uploaded an example file for that.

Yasin responded:  2018-11-21
Sorry for being annoying about that again. But at the moment FWHM really does not work to filter out cases without consecutive scans > 0 but multiple spikes within the integration range, as Skyline is not calculating the FWHM correctly in those cases (as shown in the example file above).

At the moment I have to export all chromatograms to do it in another program which takes forever as I have to monitor a lot of transitions,.. thanks a lot in advance.

Brendan MacLean responded:  2018-11-21
Hi Yasin,
Nick has left on Thanksgiving vacation (a US-only vacation). I am sure he will get back to you when he can and that he can fix any bug we may have in the FWHM calculation for your purposes if you have stated it clearly. I do note that you have posted a .skyd file, which will not help Nick. You should instead post the .sky.zip file you get from File > Share - Complete in Skyline or if that is too big to post directly to the support board, you can drop it at:


Sorry, I can't at the moment offer to fix this for you more quickly than I know Nick will once he is back, but you should give him 2 more weeks. If you expect better from us, that is a great sign of our success in supporting Skyline users.

Yasin responded:  2018-11-21
Hello Brendan,

sorry forgot that you have Thanksgiving in the US right now. I am very aware of the great support you are giving here at Skyline and really appreciate it! Just wanted to make sure that my post didn't slip through somehow.

Thanks for your great work here!

Nick Shulman responded:  2018-12-24
Hi, Yasin,

Sorry for the slow response.
In the example.sky.zip that you sent us, I see that you have some chromatograms that fluctuate between zero and non-zero, but the non-zero part forms a perfectly shaped curve.

This can happen if you have set the Mass Accuracy too small when you are also telling Skyline to use centroided data. The way the chroamtogram extraction works, if the centroided value happens to be outside of the mass accuracy window, then Skyline assigns a zero to that point in the chromatogram.
The mass accuracy is specified at:
Settings > Transition Settings > Full Scan
You have that set to 0.7ppm for both MS1 and MS2.

I cannot tell for sure that that is what is happening, since I do not have your .raw files. If you do have the raw file ("1000_C13_1.mzML"), then you can click on the chromatogram and Skyline will bring up the "Full Scan" window which will show you the scan and will have shaded areas indicating the m/z range over which Skyline was looking for intensities to sum for the chromatogram.

When you have a zig-zaggy chromatogram like that, the way that Skyline interprets the FWHM spans across all of those zig-zags. I understand that it might be more useful if the FWHM was only the distance across a single zig-zag, but it might also be less useful. We would have to do a lot of work to figure out whether we should change the behavior of this number.

If you were studying peptides, Skyline would allow you to train an mProphet model, which is able to look at more features about the peaks. I am not sure what all of those features are, but they have names like "coelution" and "shape", which I think might be helpful for what you were originally asking about.

I am not sure whether there is any way to make use of these mProphet scores in a small molecule document like you have. Maybe someone else will have better ideas.

Here's a link to the advanced peak picking tutorial, which might not be helpful to you since you do not have peptides, and therefore cannot generate decoys in order to train a model:

-- Nick