Hi Trayambak,
I just forwarded your message to Matt Chambers, the developer of IDPicker. I thought it would export BiblioSpec libraries (*.blib) directly. If it does, then those will be preferable to SpectraST libraries (*.sptxt). Also, if you are not using 3.7 or Skyline-daily, then Skyline may not support retention times in sptxt files, though it should in more recent versions of Skyline. Are you confident that you actually have RTs in the sptxt file? If so, then check you Skyline version and let us know here what it is.

Thanks for reporting your issue. I hope we can help you quickly find a way for you to get IDPicker 3 and Skyline to work together in your experiments.

--Brendan

You may also be able to build your library in Skyline from exported pepXML or mzIdentML.

HI Brendan,
Thanks for replying. Actually I have tried that option also. However, the pepXML file that I get from Myrimatch search gives this error (attached). I am trying to send you a zipped pepXML.

Thanks,
Trayambak.

Sure. This means you need to put the pepXML in a directory with your mzXML or mzML that you used as input to the search.

The pepXML you send appears to reference only 1 mzML (or mzXML) file with the root name 'ah_TB_4993_091517_WT_HR9_ECM'.

Can you still locate this file and put it in the same folder with the pepXML before you start the library build?

--Brendan

I have used "ah_TB_4993_091517_WT_HR9_ECM.raw" as initial input in Myrimatch for searching and thus generated the .pepXML file. However,now I converted the .raw to .mzML and kept it in the same directory. THis is the error I get now (attached).

Progress! I guess it has been a while since we saw pepXML from IDPicker and a Myrimatch search. We definitely used to support that during CPTAC 1 (now on CPTAC 3). Kaipo, here, will have a look at the pepXML file you supplied.

Maybe you could also upload your mzML file to http://skyline.ms/files.url

That would give Kaipo a full set of files to test with. I am sure we can get this workflow working again for you reasonably soon. Probably quicker than the sptxt, which might require a fix to IDPicker.

Thanks for your effort in finding a solution.

--Brendan

Thanks for all your Help!! I have uploaded "ah_TB_4993_091517_WT_HR9_ECM.raw" and converted (by MSCONVERT) "ah_TB_4993_091517_WT_HR9_ECM.mzML" for testing with a description "Issue Building Library from Myrimatch search".

Hope to get it resolved to perform my MS1 quantitation soon.

Hi Trayambak,
I think you may have uploaded the incorrect file - there is one named "sh_4974_TB_081817_3_WT_HR9.mzML" instead of "ah_TB_4993_091517_WT_HR9_ECM.mzML".

Thanks,
Kaipo

Extremely sorry Kaipo. I have uploaded the right .mzML , .raw and . pepXML corresponding to the same sample"ah_TB_4993_091517_WT_HR9_ECM".

Hi Trayambak,
Unfortunately it looks like we won't be able to create a spectral library from this pepXML file. When creating .blib files, we require each peptide-spectrum match to have a probability score which ranges from 0-1, but there aren't any such scores. Do you know if there is a way to get those scores into the pepXML file?

Thanks,
Kaipo

For each PSM, we get Myrimatch MVH, Myrimatch:mzFidelity and xcorr. But i do not see any probability score ranging from 0-1. I do not know how to compute those scores. Any suggestions...

Hi Trayambak,
I am going to forward this to Matt Chambers to see if it can be included in the pepXML outputs.

Thanks,
Kaipo

I've contacted Matt Chambers about this, who suggests that the MyriMatch results be run through PeptideProphet to get probability scores. Is that a possibility?

Thanks,
Kaipo

HI Kaipo, Thanks for all your help. I have never tried Peptide-prophet. I might need a little help to run through peptide-prophet and also for protein assembly protein prophet probably would require (am I right?) or just peptide prophet output would be good enough to build the library?

Skyline should be able to build a spectral library from the PeptideProphet output.

Thanks,
Kaipo

That's good news. Have you tried the pepXML to run through peptideprophet and build the library already? It would be great to have some help regarding this workflow.

Thanks again,
Trayambak.

HI Kaipo,
I tried to run peptideprophet through TPP with the pepXML file that I have from Myrimatch search. But the peptideprophet only takes .xml as input. It is not taking the pepXML. Any suggestions?

I have not used PeptideProphet before so I'm not sure - maybe it is expecting the pepXML extension to be .pep.xml rather than .pepXML? You could try renaming it and see if it accepts the input file with the alternate extension.

Thanks,
Kaipo

HI Kaipo,
I have attached a detailed ppt in this issue. If, somebody could look into the problem who has dealt TPP.

Thanks much,
Trayambak.

Note that the TPP has its own support mechanism, which you can find here:

https://groups.google.com/forum/#!forum/spctools-discuss

We are not TPP experts and you really can't expect more than that our tools handle well-formed results from the TPP. For running the TPP, you will need to consult the TPP team.

Sorry..I did not mean that. I have got this text file from uploading the .pepXML (from Myrimatch) to online TPP which has retention time details. Can it be used to generate the library?

Thanks again for all your help,
Trayambak.

Sorry for the long wait on a reply. That doesn't look like a format we recognize. It is not an XML format. So, it definitely is not pepXML despite the .pep.xml.pepXML.index ending. I think the final .index is its salient format. It would best if you you use the available TPP support to get help on finding the pepXML itself from the TPP that you would need to build your library.

One thing to keep in mind is that with pepXML, you also need the original spectral format, usually mzXML. It is not enough to have just the pepXML file (which you haven't seemed to locate yet).

Hope this helps you seek support from spctools-discuss. Thanks for your interest in using Skyline in your research.

--Brendan