Relative Protein Quantification by MS/MS Using the Tandem Mass Tag Technology

  • Loïc Dayon
  • Jean-Charles Sanchez
Part of the Methods in Molecular Biology book series (MIMB, volume 893)


The determination of protein changes related to stimuli such as pathological conditions is the core task of many proteomic studies. In the past decade, concomitantly to the increasing role of mass spectrometry (MS), several strategies have been implemented for the relative quantification of proteins with MS. Stable isotopic labels are introduced via metabolic, enzymatic, or chemical routes in different samples for their distinction during MS detection. Relative quantification is achieved by comparison of MS or tandem MS (MS/MS) signals of the differentially labeled moieties. Isobaric tagging is an elegant chemical isotope incorporation based on tags with an identical chemical structure and same total mass but with labile parts under collision-activated dissociation, the so-called reporter ions. The reporter ions are characteristic of each tag form and detected at distinct m/z. The TMT, iTRAQ, and ExacTag are examples of such technology. Experimental design, sample preparation and separation, MS acquisition parameters, and data analysis are the key steps to achieve accurate and precise quantitative measurements. We describe herein an isoelectric focusing shotgun proteomics workflow for the relative quantification of proteins in complex mixtures by MS/MS using tandem mass tags.

Key words

Proteomics Mass spectrometry Quantitation Isobaric tagging Isotope 



The authors would like to thank all colleagues involved in projects with TMTs for helpful discussions. Proteome Sciences plc. is thanked for financial support.


  1. 1.
    Gygi SP, Rist B, Gerber SA et al (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17:994–999PubMedCrossRefGoogle Scholar
  2. 2.
    Thompson A, Schafer J, Kuhn K et al (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904PubMedCrossRefGoogle Scholar
  3. 3.
    van Ulsen P, Kuhn K, Prinz T et al (2009) Identification of proteins of Neisseria meningitides induced under iron-limiting conditions using the isobaric tandem mass tag (TMT) labeling approach. Proteomics 9:1771–1781PubMedCrossRefGoogle Scholar
  4. 4.
    Dayon L, Hainard A, Licker V et al (2008) Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags. Anal Chem 80:2921–2931PubMedCrossRefGoogle Scholar
  5. 5.
    Dayon L, Pasquarello C, Hoogland C et al (2010) Combining low- and high-energy tandem mass spectra for optimized peptide quantification with isobaric tags. J Proteomics 73:769–777PubMedCrossRefGoogle Scholar
  6. 6.
    Tan HT, Tan S, Lin QS et al (2008) Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells. Mol Cell Proteomics 7:1174–1185PubMedCrossRefGoogle Scholar
  7. 7.
    Hermanson GT (2008) Bioconjugate techniques. Academic, LondonGoogle Scholar
  8. 8.
    Viner RI, Zhang T, Second T et al (2009) Quantification of post-translationally modified peptides of bovine alpha-crystallin using tandem mass tags and electron transfer dissociation. J Proteomics 72:874–885PubMedCrossRefGoogle Scholar
  9. 9.
    Byers HL, Campbell J, van Ulsen P et al (2009) Candidate verification of iron-regulated Neisseria meningitidis proteins using isotopic versions of tandem mass tags (TMT) and single reaction monitoring. J Proteomics 73:231–239PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Biomedical Proteomics Group, Department of Structural Biology and Bioinformatics, Faculty of MedicineUniversity of GenevaGenevaSwitzerland

Personalised recommendations