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Related Experiment Video

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Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer
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Peptide Labeling Using Isobaric Tagging Reagents for Quantitative Phosphoproteomics.

Lei Cheng1, Trairak Pisitkun2, Mark A Knepper3

  • 1Department of Biomedicine and Center for Interactions of Proteins in Epithelial Transport, Aarhus University, Aarhus, Denmark.

Methods in Molecular Biology (Clifton, N.J.)
|November 21, 2015
PubMed
Summary

Isobaric peptide labeling, specifically 8plex isobaric tags for relative and absolute quantitation (8plex iTRAQ), enables multiplexed quantitative phosphoproteomic analysis. This method allows assessment of peptide abundance changes in cultured cells or tissue suspensions.

Keywords:
IMACIsobaric tagsIsotopic labelingLC -MS/MSMass spectrometryMultiplexingPhosphopeptidePhosphoproteomicsReporter ionTMTiTRAQ

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Molecular Biology

Background:

  • Isobaric tagging reagents are essential for multiplexed quantitative proteomic analysis.
  • These reagents facilitate the assessment of peptide abundance changes across various experimental conditions.
  • Quantitative phosphoproteomics is crucial for understanding cellular signaling pathways.

Purpose of the Study:

  • To describe a protocol for quantitative phosphoproteomic analysis using isobaric peptide labeling.
  • To demonstrate the application of 8plex isobaric tags for relative and absolute quantitation (8plex iTRAQ) in phosphoproteomics.
  • To provide a method for analyzing cultured cells or tissue suspensions.

Main Methods:

  • Application of 8plex isobaric tags for relative and absolute quantitation (8plex iTRAQ) labeling.
  • Sample pooling followed by strong cation exchange chromatography fractionation.
  • Phosphopeptide enrichment and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

Main Results:

  • Successful quantitative phosphoproteomic analysis of cultured cells and tissue suspensions.
  • Identification and quantitation of phosphopeptides using LC-MS/MS.
  • Demonstration of the utility of 8plex iTRAQ for multiplexed phosphoproteomic studies.

Conclusions:

  • Isobaric peptide labeling with 8plex iTRAQ is a robust method for quantitative phosphoproteomics.
  • The described protocol is applicable to various biological samples, including cell cultures and tissue suspensions.
  • This approach facilitates comprehensive analysis of phosphoproteome alterations in response to different conditions.