Trouble Importing Searches from IP2

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Trouble Importing Searches from IP2 lougee  2018-06-13 09:54
 

Greetings Skyline Team,

Whenever I try to upload a spectral library from data searched on IP2 I receive the following error message. The file is saved as an MZID file and has only probability and P-value as a primary and secondary score respectively. It is a SILAC data set and we hope to utilize Skyline in analyzing the ratios. I'm not exactly sure why skyline is having an issue with the searched data. Thank you!

-Marshall

 
 
Brendan MacLean responded:  2018-06-13 10:43

Hi Marshall,
Unfortunately, mzIdentML files do not contain spectra. So, in order to build a spectral library from them, you will need to also make data files with spectra in them matching the source files listed in the mzIndentML file. The error message means what it says. The library builder is looking for one of 3 possible matching file types (mzML, mzXML or MGF) to go with your mzIdentML search results. If you have any of these, you can put them in the same folder as the mzIdentML file, and that should solve the problem.

If you do not have any of these, we'll need to understand your search pipeline better and how you ended up with these mzIdentML files. I can't say for sure whether we have ever seen IP2 output, but at least the error message you sent is not complaining about not being able to understand the score types in your mzIdentML.

We'll try to help you get this working. Let's start with you putting the spectrum files in the same folder with the mzIdentML.

Thanks for your interest in using Skyline in your research.

--Brendan

 
lougee responded:  2018-06-13 11:41

Hi Brendan,

Thanks for getting back to me so fast! I am trying to use the "Import DDA Peptide Search" setup wizard. I have my raw file converted to a mzXML file as well, however, when I try to add the two files to build my library it says that "mzXML is not a valid library format." Should I try converting to MGF and seeing if this works? Just to further clarify. I use IP2 to search my data, I then export this as an mzIdentML file. I also convert the matching raw file to mzXML format. However, when I try to add both of these to build my spectral library it states that mzXML is not a valid library input file.

Thanks!

-Marshall

 
Brendan MacLean responded:  2018-06-13 11:59

Hi Marshall,
You want to continue specifying the mzIdentML file you started with, but make sure your mzXML files are in the same folder on your disk drive with that mzIdentML file. That should at least get you past the error message you sent originally.

--Brendan

 
lougee responded:  2018-06-13 13:20

Brendan,

Okay, so that seems to have worked but now it is simply returning the same error message with the file name and an H in front of it. I.e. if the file name is XYZ it finds XYZ but now says HXYZ could not be found in the directory. This seems a little strange to me. Perhaps this is something going on with IP2 and not skyline now. It may be an artifact from IP2 script in the mzIdentML file that is confusing skyline.

-Marshall

 
Matt Chambers responded:  2018-06-25 11:09

Hi Marshall,

I will try to dig into this soon. Can you upload an mzIdentML so I can see what's in there to work with?

Thanks,
-Matt

 
lougee responded:  2018-06-25 11:58

Hey Matt,

Thanks for getting back. We were actually able to fix the issue. It was a problem with how the mzIdentml files were created for SILAC experiments in IP2. It creates two separate libraries, one for heavy and one for light, and skyline did not recognize the heavy library-as the raw file name is changed slightly in the mzIdentml file (i.e. skyline tries to find a raw file that does not exist).

We talked with the people at IP2 and they were able to implement a fix!! You guys have been very helpful though, and we love how skyline is working so far-getting great data back. We will certainly be using it in the future!

Thank you,

-Marshall