Peptide Labeling Using Isobaric Tagging Reagents for Quantitative Phosphoproteomics

Methods Mol Biol. 2016;1355:53-70. doi: 10.1007/978-1-4939-3049-4_4.


Isobaric tagging reagents have become an invaluable tool for multiplexed quantitative proteomic analysis. These reagents can label multiple, distinct peptide samples from virtually any source material (e.g., tissue, cell line, purified proteins), allowing users the opportunity to assess changes in peptide abundances across many different time points or experimental conditions. Here, we describe the application of isobaric peptide labeling, specifically 8plex isobaric tags for relative and absolute quantitation (8plex iTRAQ), for quantitative phosphoproteomic analysis of cultured cells or tissue suspensions. For this particular protocol, labeled samples are pooled, fractionated by strong cation exchange chromatography, enriched for phosphopeptides, and analyzed by tandem mass spectrometry (LC-MS/MS) for both peptide identification and quantitation.

Keywords: IMAC; Isobaric tags; Isotopic labeling; LC -MS/MS; Mass spectrometry; Multiplexing; Phosphopeptide; Phosphoproteomics; Reporter ion; TMT; iTRAQ.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cation Exchange Resins
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Computational Biology
  • Databases, Protein
  • Humans
  • Isotope Labeling*
  • Phosphopeptides / analysis*
  • Phosphopeptides / chemistry
  • Phosphopeptides / metabolism
  • Phosphorylation
  • Protein Processing, Post-Translational
  • Proteomics / methods*
  • Tandem Mass Spectrometry
  • Workflow


  • Cation Exchange Resins
  • Phosphopeptides