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MIDAS Libraries Questions roman sakson  2018-10-14 11:07
 

Hello Skyline team,

thank you very much for the great piece of software that you are developing and maintaining! In our core facility for proteomics in Heidelberg, Germany, I work with a QTrap 5500 and we often use the Sciex MIDAS workflow. Some time ago, you have implemented more support for this in Skyline, which is great. However, I have two general question:

When I import a wiff-file containing MRM traces as well as MS/MS spectra into Skyline, as it is the case for MIDAS experiments, I see the usual MRM XICs and also vertical blue lines with retention times over them. I believe, that this lines indicate the timepoints at which MS/MS spectra were acquired in my wiff-file. Importantly, there is no "ID" word on top of those lines yet, since no "real" spectral library containing a database results output like a dat-file from Mascot is present in the Skyline document at that stage. However, since there is now a "midas" library created by Skyline automatically, I still get to see all the imported MS/MS spectra under "Library Match" with a more or less long list of all those spectra with a corresponding RT in brackets offered to me as a dropdown menu. I appreciate that but I have realized that there is also the peptide sequence and a charge state displayed on top of the library match, just as how it would look like for a "real" spectral library match (see first screenshot). At that point, Skyline probably just imports the peptide sequence from the target list, since it has no database results available yet, and I don't really know what amino acid sequence corresponds to the MS/MS spectrum. Am I missing something here?

The second question concerns the new tab "Library Runs" which I can find under "Manage Results". I believe, that there is an entry created for each MIDAS-experiment that I import (see second screenshot). While the "midas" library is automatically created by Skyline in the same directory in which the Skyline-file itself lives, the path for the Library Run seems to point to the folder where my original wiff-files are stored. I don't have any particular issues with that but would just like to understand whether my wiff-files are now somehow connected to my Skyline-files? What would happen if I made use of the "Remove corresponding runs" check box?

Kind regards,

Roman
 
 
roman sakson responded:  2018-10-22 08:01
Title: MIDAS Libraries Questions Repeat

Dear Skyline Team,

I just wanted to make sure that my question above was not accidentially overlooked, since it has been over a week by now and you normally respond within a couple of hours :).

Kind regards,
Roman
 
Nick Shulman responded:  2018-10-22 12:55
When Skyline encounters MS2 spectra in a WIFF file, Skyline creates a .midas file which contains those spectra. Skyline also remembers which peptide Skyline was extracting chromatograms for when it encountered the spectrum.

When Skyline shows you a spectrum from a MIDAS file, Skyline annotates the MS2 peaks with the names ("y7++", etc) of the fragment ions that could have produced them. These annotations are not stored in the .midas file-- the spectrum in the .midas file is just a list of mzs and intensities. Skyline does use the peptide sequence that is stored in the .midas file in order to figure out how to annotate the fragment ions. However, Skyline does not really need that-- Skyline knows which peptide you have selected, and so Skyline can annotate any spectrum as if it was produced by that peptide. If you see that very few of the peaks of the spectrum were annotated with the name of a fragment ion, then it is likely that the spectrum was not produced by fragmenting that peptide.

In the "Manage Results" dialog, the "Remove corresponding library runs" is referring to removing data from the .midas file. Skyline would never try to delete your original .wiff files.

I hope this helps, but I'm not sure if I understood your question.
-- Nick
 
roman sakson responded:  2018-10-22 13:19
Title: Further MIDAS Libraries Questions
Hi Nick,

thank you for your response and yes, it does help me! I am just trying to understand the features around the MIDAS workflow in Skyline a bit better since I am working at the establishment of a robust MRM assay development workflow. At the moment we combine the midas spectral library with a "normal" spectral library from a mascot dat-file to check for IDs, so I guess that we are fine there. I have realized that a midas library can be filtered via peptide library settings to create another spectral library, could you please explain to me what happens there?

Another issue that I have noticed is that if I have more then one sample run within the same wiff-file (which we have a lot), then the midas library is created only for the first sample in the wiff file and not for subsequent ones. Is this a known effect? I work with Skyline, not the daily version. I know that there are some effects like that related to Panorama and that there the AutoQC-based upload of more then one sample per wiff-file works in the daily version of Skyline but not for the regular release.

Thanks a lot in advance!

Roman
 
Nick Shulman responded:  2018-10-22 13:47
I am not sure whether we ever tested the MIDAS library feature with multi sample .wiff files. What you are describing sounds like a bug in Skyline.

Can you send me an example of a multi-sample .wiff file (and .wiff.scan) with MIDAS scans as well as your Skyline document (.sky.zip)?

If those files are less than 50MB, then you can attach them to this support request. Otherwise, you can upload them here:
https://skyline.ms/files.url
 
roman sakson responded:  2018-10-26 14:26
Hi Nick,

I am sorry for the belated reply, we have had some issues with our instruments... Please, find the requested files attached. The .wiff file contains two samples with MIDAS spectra of two peptides in each sample, respectively (different charge states per peptide also included). Peptides EDLAD... and AVT... were in the first sample and MIDAS spectra are extracted in Skyline just fine. Peptides HVV... and IQIV... were in the second sample and there are no MIDAS spectra extracted (both samples were imported simultaneously as two single-injection replicates). It would be great if we could have a solution for that, since we would like to be able to group our runs as multi sample .wiff files (which then can be all converted into a single .mgf file for Mascot instead of converting each sample separately).

Roman