SpectraST fail to open my files in windows and in Linux as well

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Hugo Amedei

Apr 13, 2021, 11:27:31 AM4/13/21
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Hi everyone,
how are you? I am PhD student from Göttingen, DE.
I am using Spectrast to create my library  from Sequence Search Results (comet and X!tandem nature protocol Olga T Schubert, 2015).
I did a SEC fractionation MS experimient- 53 fractions- I measured with Q-exactive DDA 12 more intense peptides. And I would like to use my library for SWATH (I will also use Q-exactive for DIA).
The bioinformatic workflow was// seach comet - seach xtamdem// Peptideprophet (hier I got only two files)// then iprophet (hier I got 1 file combined)// and now I want to create my library by SpectraST I got the error when spectra ST goes to the files to extract the peptides, it can not open the files!! (see picture attached).
     This happend to me in DOS-windows, but in Ubuntu 20.04 as well.
Do you know what could be the mistake?.
 I used Ms convert- I try Creating new mzXML files and I got allways the same error/ My Iprophet file input weigh 550 mb. then files peptideprophet search 1 weight 500 mb and search 2 1,6Gb. the files have of peptides and 4000 proteins predicted (PEP3D). But spectral library is empty.
Thank you for your support, I really try everything I could to make it works.

   Hugo Amedei.


David Shteynberg

Apr 13, 2021, 2:29:42 PM4/13/21
to spctools-discuss
Hello Hugo,

It appears from the error message that SpectraST is unable to find any PSMs having probabilities above 50% in your iProphet file.   Can you verify that you have PSMs with iProphet probabilities above 50% in your pepXML file?  If not I would look in the upstream analysis for the error.  Can you check your database and search parameters and make sure PeptideProphet can find a population of correctly identified PSMs in the search engine results?  If your search parameters and database look correct, the issue could be with the data itself.

Thank you,

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