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DIGE Analysis of Immunodepleted Plasma.

Paul Dowling1, Kay Ohlendieck2

  • 1Department of Biology, Maynooth University, National University of Ireland, Maynooth, Co. Kildare, Ireland. paul.dowling@nuim.ie.

Methods in Molecular Biology (Clifton, N.J.)
|October 12, 2017
PubMed
Summary

This study enhances biomarker discovery by removing abundant proteins from plasma using immunodepletion. This technique, combined with difference gel electrophoresis (DIGE), improves the detection of low-abundance proteins for disease research.

Keywords:
AntibodiesImmunodepletionLow abundantPlasmaSerum

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Area of Science:

  • Proteomics
  • Biomarker Discovery
  • Analytical Chemistry

Background:

  • Biofluid proteomes exhibit a vast dynamic range, with high-abundance proteins masking low-abundance disease biomarkers.
  • Difference gel electrophoresis (DIGE) is a powerful quantitative proteomics technique, but its effectiveness is limited by the dynamic range of samples.
  • Detecting and identifying medium- and low-abundance proteins is crucial for comprehensive biomarker strategies.

Purpose of the Study:

  • To detail the upstream immunodepletion of high-abundance proteins from plasma samples.
  • To demonstrate the subsequent analysis of depleted samples using difference gel electrophoresis (DIGE).
  • To highlight the combined approach for enhancing the detection and identification of disease-specific biomarkers.

Main Methods:

  • Upstream immunodepletion of high-abundance proteins from plasma using antibody columns.
  • Quantitative proteomic analysis of depleted samples via difference gel electrophoresis (DIGE).
  • Integration of immunodepletion with DIGE for comprehensive biofluid proteome analysis.

Main Results:

  • Immunodepletion successfully reduces the dynamic range of plasma proteomes.
  • Enhanced detection and quantification of medium- and low-abundance proteins are achieved.
  • The combined immunodepletion-DIGE method broadens proteomic coverage and facilitates biomarker identification.

Conclusions:

  • Combining immunodepletion with DIGE is an effective strategy for biomarker discovery in complex biofluids.
  • This approach significantly improves the ability to identify disease-specific proteins.
  • The method provides a robust and reproducible fractionation and quantification technique for proteomic studies.