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

Anal Chem. 2010 Aug 1;82(15):6549-58. doi: 10.1021/ac100890k.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • HeLa Cells
  • Humans
  • Peptides / chemistry*
  • Piperazines / chemistry*
  • Piperidines / chemistry*
  • Proteomics / methods*
  • Succinimides / chemistry*


  • Peptides
  • Piperazines
  • Piperidines
  • Succinimides