Isobaric labeling-based relative quantification in shotgun proteomics

J Proteome Res. 2014 Dec 5;13(12):5293-309. doi: 10.1021/pr500880b. Epub 2014 Nov 4.


Mass spectrometry plays a key role in relative quantitative comparisons of proteins in order to understand their functional role in biological systems upon perturbation. In this review, we review studies that examine different aspects of isobaric labeling-based relative quantification for shotgun proteomic analysis. In particular, we focus on different types of isobaric reagents and their reaction chemistry (e.g., amine-, carbonyl-, and sulfhydryl-reactive). Various factors, such as ratio compression, reporter ion dynamic range, and others, cause an underestimation of changes in relative abundance of proteins across samples, undermining the ability of the isobaric labeling approach to be truly quantitative. These factors that affect quantification and the suggested combinations of experimental design and optimal data acquisition methods to increase the precision and accuracy of the measurements will be discussed. Finally, the extended application of isobaric labeling-based approach in hyperplexing strategy, targeted quantification, and phosphopeptide analysis are also examined.

Keywords: TMT; iTRAQ; isobaric labeling; isobaric tags; isobaric tags for relative and absolute quantification; mass spectrometry; quantitative proteomics; tandem mass tags.

Publication types

  • Research Support, N.I.H., Extramural
  • Review

MeSH terms

  • Amines / chemistry
  • Amines / metabolism
  • Isotope Labeling / methods*
  • Mass Spectrometry / methods*
  • Phosphopeptides / analysis
  • Phosphopeptides / metabolism
  • Protein Carbonylation
  • Proteome / analysis*
  • Proteome / chemistry
  • Proteome / metabolism
  • Proteomics / methods*
  • Reproducibility of Results
  • Sulfhydryl Compounds / chemistry
  • Sulfhydryl Compounds / metabolism


  • Amines
  • Phosphopeptides
  • Proteome
  • Sulfhydryl Compounds