title: Peptide labeling with isobaric tags yields higher identification rates using iTRAQ 4-plex compared to TMT 6-plex and iTRAQ 8-plex on LTQ Orbitrap.
abstract: Peptide labeling with isobaric tags has become a popular technique in quantitative shotgun proteomics. Using two different samples viz. a protein mixture and HeLa extracts, we show that three commercially available isobaric tags differ with regard to peptide identification rates: The number of identified proteins and peptides was largest with iTRAQ 4-plex, followed by TMT 6-plex, and smallest with iTRAQ 8-plex. In all experiments, we employed a previously described method where two scans were acquired for each precursor on an LTQ Orbitrap: A CID scan under standard settings for identification, and a HCD scan for quantification. The observed differences in identification rates were similar when data was searched with either Mascot or Sequest. We consider these findings to be the result of a combination of several factors, most notably prominent ions in CID spectra as a consequence of loss of fragments of the label tag from precursor ions. These fragment ions cannot be explained by current search engines and were observed to have a negative impact on peptide scores.
authors.affiliation: Christian Doppler Laboratory for Proteome Analysis, University of Vienna, Austria. pichler@imp.univie.ac.at
authors.forename: Peter
authors.initials: P
authors.lastname: Pichler
authors.forename: Thomas
authors.initials: T
authors.lastname: Köcher
authors.forename: Johann
authors.initials: J
authors.lastname: Holzmann
authors.forename: Michael
authors.initials: M
authors.lastname: Mazanek
authors.forename: Thomas
authors.initials: T
authors.lastname: Taus
authors.forename: Gustav
authors.initials: G
authors.lastname: Ammerer
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
mesh_terms: D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D010879:Piperazines / Q000737:chemistry*; D010880:Piperidines / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D013388:Succinimides / Q000737:chemistry*
chemical_list: D010455:Peptides; D010879:Piperazines; D010880:Piperidines; D013388:Succinimides