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 spectral libraries and extracted chromatograms.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url

I am not sure I understand what sort of peptide search results you have but after we see your files I imagine your question will make more sense.
--Nick

Hi Nick,

Thanks for your help! I’ve uploaded the requested file to the "File Sharing Folder for Skyline Support" under the name "ArdecheMRM1STPro.sky.zip." :)

Best,
Amir

It looks like your peptide search engine matched spectra to "ALLNAIGNLELTGAFAEALK" in several of your replicate.
In your first screenshot, Skyline is drawing dozens of ID lines on the chromatogram graph, which means that there were many peptide spectrum matches (PSMs) for that peptide in that file.
In your second screenshot, there is a single ID line which means that there was one PSM for that peptide in that file.
The reason that the ID line is red in that second screenshot is because that is the spectrum which is being shown in the "Library Match" window.
There is a dropdown at the top of the Library Match window where you could choose a spectrum from a different replicate, and, if you chose something else the color of that ID line would change.

I do not understand what you mean when you say "none of the other peptides in this sample are detected, and this same peptide isn't identified in the other replicates". Every peptide has been detected in several replicates.

If you want to see the complete list of peptides you can go to:
View > Spectral Libraries
If you select a peptide in the Library Explorer, you can use the "File" dropdown below the spectrum to see which other files the peptide was detected in.

I see that at "Settings > Transition Settings > Full Scan" you have the MS/MS Acquisition Method set to "DDA".
When Skyline extracts chromatograms from DDA data, the chromatograms usually look very strange with long straight lines connecting the places where a matching precursor happens to have been selected by the mass spectrometer for fragmentation. For this reason, DDA MS2 chromatograms in Skyline are not useful for quantification and, so, Skyline displays the MS2 chromatograms as dotted lines.
Another thing that you might have noticed is that if you do not also tell Skyline to extract MS1 chromatograms in addition to the MS2 chromatograms, you do not get any MS2 DDA chromatograms. It looks like you might have already worked around this by setting the "MS1 Isotope peaks included" at "Settings > Transition Settings > Full Scan" to "Count".

Skyline is primarily a tool for extracting chromatograms. It is possible to use Skyline to look at DDA peptide search results, but the features that Skyline has in this area only exist because they are also useful for extracting chromatograms. If all you want to do is look at your DDA search results, there are probably better tools, but I am not sure what they are.

I see that your spectral library was created from .mzid files, so I am guessing the peptide search engine that you used was MSGF+, but it might have been something else.

You might find some helpful information in one of Skyline's tutorials in the "Full-Scan Acquisition Data" section:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorials
-- Nick

Hi Nick,

Thank you for your previous response. I've been working through Skyline tutorials and support sessions to analyze MRM data generated by an old Ion Trap mass analyzer (HCT Bruker), which limits my software options due to the data format. Based on my research, I applied the "MS1 Full-Scan Filtering" tutorial to work with DDA data (these data are not presented here). After running a DDA measurement, I adjusted the settings to view the results in Full Scan mode. Finally, I could analysis my data, but I wanted to perform Target proteomics. However, the only feasible way to conduct targeted proteomics on our instrument is through MRM.

In my experiment, I selected three peptides from one protein:

K.YHAEFTPVFSPER
R.DSLLINWNATYDIYEK
R.ALLNAIGNLELTGAFAEALK
Currently, only the transitions of the peptide "R.ALLNAIGNLELTGAFAEALK.N" (mass 1015.07++) display green ticks, with a yellow triangle indicating that at least half of the transitions contribute to a co-eluting peak. This is a breakthrough, as I've been struggling for months to achieve this. In the "Peak Area Percentage" graph, this peptide shows a bar, but the other peptides do not have any bars, and all transitions are red.

I’d like to achieve consistent peak integration and peak area visualization across all replicates and peptides, as I currently only see it for the last peptide. Could you provide guidance on how to replicate this success for all replicates and peptides? This would alleviate a great deal of pressure, as I have exhausted available resources and tutorials without finding a solution.

Thank you very much for your assistance!

Best,
Amir

The red, yellow and green symbols next to the items in the Targets tree provide Skyline's opinion of the chromatogram peak shape.
There is a bit of explanation of what those symbols mean here:
https://skyline.ms/announcements/home/support/thread.view?rowId=64988

I believe the reason that everything has a red symbol next to it is that Skyline thought this was DDA data and Skyline did not do any peak detection because Skyline never tries to detect peaks in DDA data.
If you want Skyline to do peak detection you could go to "Settings > Transition Settings > Full Scan" and change the MS/MS filtering Acquisition method to "PRM".
After you do that, you should go to "Edit > Manage Results" and use the "Rescore" button.
Skyline will then perform peak detection on the chromatograms that have already been extracted and more transitions in the Targets tree will get green symbols next to them.

The reason that there was one replicate and one peptide with a green symbol next to it in the document that you sent us is that a user manually set the peak boundaries for that one item. If a user has chosen a peak, then Skyline always displays symbols in the Targets tree indicating that it is a good-looking peak.

I might be able to give you better advice if you send us some of your raw files. I see that the chromatograms were extracted from .mzML files. You could either send us some of those .mzML files and/or you could zip up the original Bruker .d folders.
-- Nick

Hi Nick,

Thank you very much for your guidance. Switching the MS/MS filtering Acquisition method to PRM and using the Rescore button worked perfectly! I now see more transitions with green symbols, and the peak detection is much improved. I’ve created a zip file (241022MRM26.zip) with six replicates of the raw .d data, which I’m uploading now to the "File Sharing Folder for Skyline Support". Due to the large file sizes, I do not upload .mzML files, but I’ve attached a screenshot of the MSConvert settings I used when I initially converted these data.
Please let me know if you need further details or adjustments on my end!

Thanks so much for your continued assistance.

Best regards,
Amir

Thanks for uploading those files. I will try to take a look at them soon.
By the way, it usually is not necessary to convert Bruker .d folders to mzML before importing into Skyline.
MSConvert and Skyline understand the same file formats.
--Nick

Thank you for the tip about importing Bruker .d folders directly into Skyline! I wanted to mention that, because our mass analyzer is quite old and generates data in the .yep format, converting to .mzML has been the only way I’ve found to make these files compatible with analysis software. This is what I’ve figured out so far—though I could be mistaken, I just wanted to keep you informed.

I really appreciate you taking the time to review my files and helping me through this! :)

Best regards,
Amir

I see that this was a PRM experiment where the mass spectrometer was told to repeatedly select the same precursor m/z values for fragmentation. Skyline will do a good job extracting MS2 chromatograms for this type of data.

It seems that I was mistaken about Skyline being able to read this type of data. In order for Skyline to recognize Bruker data as being readable, the .d folder must contain a file with the name "analysis.baf", "analysis.tdf" or "analysis.tsf". Your folders contain "analysis.yep" files which MSConvert is clearly able to handle. I will ask around and we will probably be able to fix this in Skyline soon.
-- Nick

Hi Nick,

Thank you for clarifying that this was indeed a PRM experiment and for the information on Skyline’s requirements for Bruker data files. It’s great to hear that you’re considering adding support for the “analysis.yep” format in Skyline, as this would simplify the workflow considerably for users with older Bruker instruments.

I appreciate you checking into this and for all your assistance! :)

Best regards,
Amir