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.
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.
Pichler
Peter
P
Christian Doppler Laboratory for Proteome Analysis, University of Vienna, Austria. pichler@imp.univie.ac.at
Köcher
Thomas
T
Holzmann
Johann
J
Mazanek
Michael
M
Taus
Thomas
T
Ammerer
Gustav
G
Mechtler
Karl
K
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*
D010455:Peptides; D010879:Piperazines; D010880:Piperidines; D013388:Succinimides