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

• Published in: Analytical chemistry (Washington) Vol. 82; no. 15; pp. 6549 - 6558
• Main Authors: Pichler, Peter, Köcher, Thomas, Holzmann, Johann, Mazanek, Michael, Taus, Thomas, Ammerer, Gustav, Mechtler, Karl
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-08-2010
• Subjects: Analytical chemistry; Biological and medical sciences; Comparative analysis; Diverse techniques; Fundamental and applied biological sciences. Psychology; HeLa Cells; Humans; Ions; Molecular and cellular biology; Peptides; Peptides - chemistry; Piperazines - chemistry; Piperidines - chemistry; Proteins; Proteomics; Proteomics - methods; Succinimides - chemistry; Collisional activation; Peptides; Sample; Standard; Identification; Method; Precursor; Mixture; Protein; Sample preparation; Proteomics; Yield; Technique; Quantitative analysis
• ISSN: 0003-2700; 1520-6882
• 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.