Interference peaks and peak tailing thetrung126829412  2024-01-25 07:26

I ran DDA samples to check a few synthetic peptide pairs (light and heavy, run separately) for purity before spiking the heavy peptides in my biological samples. Based on the extracted chromatograms shown in the skyline, some of the peptides are not so pure (95% purity from the manufacturer). These strong interference peaks (both MS1 and MS2 levels) are however still distinguishable thanks to the intended synthetic peaks of the heavy or light synthetic peptides.

However, when I spiked the heavy peptides in my biological samples (PRM experiments), due to the low signal intensities of the endogenous peptides, the peaks were quite difficult for me to distinguish.
For example, in peptide 1, the peaks y7 and y6 of the endogenous peptide have some really strong interference peaks, I could of course remove these peaks and still use the rest of the peaks for quantification. However, I am afraid the other three peaks might not be enough to confirm detection of the peptide 1.
For peptide 2, there are only three product peaks for both the endogenous and the heavy spiked-in peptide. It looks quite similar to the DDA run of the peptide 2. The interference peaks are very strong and eluted later than the intended peaks. So, it could be safe to conclude that the endogenous peptide was not detected in this sample.
For peptide 3, there are 4 product peaks for both the endogenous and the heavy spiked-in peptide. It looks quite similar to the DDA run of the peptide 3. However, in this case, the intended peaks have much higher intensity values than the interference peaks. The interference peaks eluted later than the intended peaks and they almost looked like tails. So, I think I could conclude that the endogenous peptide was detected in this sample but I am not 100% sure.
Could you please help me take a look at these pictures and let me know what you think? I appreciate your help.

Nick Shulman responded:  2024-01-25 07:57
I am not sure that I understand what you are asking but here is some information that might be helpful:

1. If you have specified an "Internal standard type" at "Settings > Peptide Settings > Modifications" then Skyline will do peak detection by looking at the internal standard's chromatograms. The chromatograms from the endogenous transitions will not really affect Skyline's decision about which is the correct peak, although they might have a small effect on the exact placement of the boundaries as Skyline extends the peak boundaries outwards to make sure they encompass the entire peak.
Skyline assumes that the internal standard will be much easier to detect than the endogenous peptide.
If your heavy peptides are not that easy to detect, then you should go to "Settings > Peptide Settings > Modifications" and choose "none" as the "Internal standard type".

2. If you have a Transition that has interference in it, but which you still want to use for peak detection, then you should mark the Transition as non-quantitative. To do that, you can right-click on the Transition in the Targets tree and uncheck the menu item "Quantitative".
When a Transition has been marked as non-quantitative its area will not be included in the Total Area value reported for the Precursor.

3. If you want more information about why Skyline chose a particular peak you should use the menu item "View > Other Grids > Candidate Peaks". The Candidate Peaks window shows all of the peaks that Skyline detected in that replicate along with the various scores that they were assigned. Skyline chooses the candidate peak with the highest combined score.

4. When you have an internal standard, we don't usually talk about whether a peptide was "detected" in the sample. The peptide is always detected in the sense that Skyline was able to set the integration boundaries based on the heavy peptide's chromatograms. The number you would report in your results would be the ratio of the light peptide's signal to the heavy peptide's signal and that might be a small number or a big number. If you would like to compare peak areas between groups of replicates you should look at the Group Comparison tutorial:

We might be able to give you a better answer if 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.
You can upload larger files here:
-- Nick
thetrung126829412 responded:  2024-01-25 09:34
Hi Nick,
Thank you so much for your quick response.
1. I have done this and Skyline has picked the transitions based on the heavy standard. This mean that Skyline also selected transitions for the endogenous peptide based on the info from the heavy standard. However, in doing so, it selected the transitions that have interference right before or after the placement of the boundaries (Peptide 1 and 3 PRM). That's why I am just confused. Could I trust that I have detected the endogenous peptide or not when there are interferences like this?
2. This is very good to know. Thanks so much. Take peptide 3-PRM for example. all the transitions have interference and if I don't select them for quantification, I will have nothing for quantification.
3. Great, thanks. This is nice to know.
4. Thanks for the clarification. I hope you understand my concern as I have mentioned in point 1. When the selected transitions have interference in them. How can I really trust that these are the right ones?
I will upload the skyline files.
Thank you so much for helping me again.
Trung Tran
thetrung126829412 responded:  2024-01-25 09:42
Hi Nick,
I actually will have some issues with data security. Is there another way for me to send you the skyline documents? Thanks