Post-acquisition ETD spectral processing for increased peptide identifications

  • David M. Good
  • Craig D. Wenger
  • Graeme C. McAlister
  • Dina L. Bai
  • Donald F. Hunt
  • Joshua J. Coon
Focus: The Orbitrap


Tandem mass spectra (MS/MS) produced using electron transfer dissociation (ETD) differ from those derived from collision-activated dissociation (CAD) in several important ways. Foremost, the predominant fragment ion series are different: c- and z·-type ions are favored in ETD spectra while b- and y-type ions comprise the bulk of the fragments in CAD spectra. Additionally, ETD spectra possess charge-reduced precursors and unique neutral losses. Most database search algorithms were designed to analyze CAD spectra, and have only recently been adapted to accommodate c- and z·-type ions; therefore, inclusion of these additional spectral features can hinder identification, leading to lower confidence scores and decreased sensitivity. Because of this, it is important to pre-process spectral data before submission to a database search to remove those features that cause complications. Here, we demonstrate the effects of removing these features on the number of unique peptide identifications at a 1% false discovery rate (FDR) using the open mass spectrometry search algorithm (OMSSA). When analyzing two biologic replicates of a yeast protein extract in three total analyses, the number of unique identifications with a ∼1% FDR increased from 4611 to 5931 upon spectral pre-processing—an increase of ∼28. 6%. We outline the most effective pre-processing methods, and provide free software containing these algorithms.


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Copyright information

© American Society for Mass Spectrometry 2009

Authors and Affiliations

  • David M. Good
    • 1
  • Craig D. Wenger
    • 1
  • Graeme C. McAlister
    • 1
  • Dina L. Bai
    • 2
  • Donald F. Hunt
    • 2
    • 3
  • Joshua J. Coon
    • 1
    • 4
  1. 1.Department of ChemistryUniversity of WisconsinMadisonUSA
  2. 2.Department of ChemistryUniversity of VirginiaCharlottesvilleUSA
  3. 3.Department of PathologyUniversity of VirginiaCharlottesvilleUSA
  4. 4.Department of Biomolecular ChemistryUniversity of WisconsinMadisonUSA

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