Skyline builds spectral libraries using a separate program called BiblioSpec, which has two main components. BlibBuild is called to build the redundant library, which is then filtered by BlibFilter to create the non-redundant library. The BlibBuild page contains information on the various search engines that are supported, along with information about their respective file formats and the scores used with the cut-off value specified in Skyline.

BlibFilter chooses the best spectrum within a group by simply using the one with the best score. If there are multiple spectra tied for the best score, the one with the highest TIC is selected. In the past, BlibFilter chose the spectrum with the highest average dot product when compared to all other spectra within the same group, but this method occasionally produced poor results. A similar method, computing a consensus spectrum and its dot product against the related spectra, also produced inferior results as it sometimes resulted in high-noise spectra being chosen.

Skyline with BiblioSpec supports building libraries from the following peptide spectrum matching pipeline outputs:

  • Mascot (DAT)
  • ByOnic (mzIdentML / MGF)
  • Comet (sqt / ms2, pepXML / mzXML)
  • Myrimatch / IDPicker (mzIdentML / mzML)
  • MaxQuant Andromeda (msms.txt with modifications.xml)
  • Morpheus (pepXML / mzXML)
  • MSGF+ (mzIdentML / mzML)
  • OMSSA (pepXML / mzXML)
  • Peaks (pepXML / mzXML)
  • PeptideShaker (mzIdentML)
  • PRIDE XML
  • Protein Pilot (group or group.xml)
  • Protein Prospector (pepXML / mzXML)
  • Proteome Discoverer (MSF / pdResult)
  • Scaffold (mzIdentML / MGF)
  • Spectrum Mill (pepXML / mzXML)
  • TPP (pepXML / mzXML)
  • X! Tandem (xml output)
  • PLGS - Waters MSe (final_fragment.csv)
  • Custom (SSL)


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