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iTRAQ-Based Shotgun Proteomics Approach for Relative Protein Quantification.

Erika Velásquez Núñez1, Gilberto Barbosa Domont1, Fábio César Sousa Nogueira2

  • 1Laboratory of Protein Chemistry - Proteomics Unit, Chemistry Institute, Federal University of Rio de Janeiro, Bloco A, Lab 543, Avenida Athos da Silveira Ramos 149, Cidade Universitária, 21941-909, Rio de Janeiro, RJ, Brazil.

Methods in Molecular Biology (Clifton, N.J.)
|November 30, 2016
PubMed
Summary
This summary is machine-generated.

This study details a method for shotgun proteomics using Isobaric tagging for relative and absolute quantitation (iTRAQ) 4-plex labeling. The protocol enables precise protein quantification in human and plant samples, aiding functional role understanding.

Keywords:
Animal tissuesMass spectrometryPlant tissuesQuantitative proteomicsStable isotope labelingiTRAQ

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Biochemistry

Background:

  • Shotgun proteomics is vital for quantifying proteins and understanding their functions under varying conditions.
  • Isobaric tagging for relative and absolute quantitation (iTRAQ) mass spectrometry allows simultaneous analysis of up to eight proteomes via peptide pre-labeling.

Purpose of the Study:

  • To present a detailed protocol for sample preparation and iTRAQ 4-plex labeling for relative protein quantification.
  • To offer strategies for peptide fractionation post-iTRAQ labeling for enhanced analysis.

Main Methods:

  • Sample preparation for human and plant tissues.
  • iTRAQ 4-plex labeling of peptides.
  • Two distinct peptide fractionation techniques following labeling.

Main Results:

  • A comprehensive protocol for iTRAQ 4-plex labeling is established.
  • Effective peptide fractionation strategies are demonstrated for improved quantitative proteomics.

Conclusions:

  • The described protocol facilitates robust relative protein quantification across multiple samples.
  • This methodology enhances the application of iTRAQ mass spectrometry in comparative biological studies.