Post-acquisition ETD spectral processing for increased peptide identifications.
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 approximately 1% FDR increased from 4611 to 5931 upon spectral pre-processing--an increase of approximately 28.6%. We outline the most effective pre-processing methods, and provide free software containing these algorithms.
Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
David M
DM
Good
Craig D
CD
Wenger
Graeme C
GC
McAlister
Dina L
DL
Bai
Donald F
DF
Hunt
Joshua J
JJ
Coon
D000465:Algorithms; D013058:Mass Spectrometry / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D012984:Software
D010455:Peptides