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Glycopeptide Capture for Cell Surface Proteomics
10:11

Glycopeptide Capture for Cell Surface Proteomics

Published on: May 9, 2014

Proteomic approach to the identification of cell membrane proteins

H Watarai1, Y Inagaki, N Kubota

  • 1Pharmaceutical Research Laboratory, Kirin Brewery Co. Ltd., Takasaki, Gunma, Japan.

Electrophoresis
|February 16, 2000
PubMed

Insights

Comparing cell disruption methods for U937 cells, nitrogen cavitation yielded purer plasma membrane protein fractions than Dounce homogenization for proteomic analysis.

Area of Science:

  • Cell Biology
  • Proteomics
  • Biochemistry

Background:

  • Plasma membrane proteins are crucial for cellular functions.
  • Accurate isolation of plasma membranes is essential for proteomic studies.
  • U937 cells are a common model for monocyte research.

Purpose of the Study:

  • To compare two cell disruption methods for isolating plasma membrane proteins from U937 cells.
  • To evaluate the purity of isolated fractions using proteomic techniques.
  • To optimize methods for plasma membrane protein analysis.

Main Methods:

  • Human monocyte-derived U937 cells were used.
  • Two cell disruption techniques were compared: Dounce homogenization and nitrogen cavitation.
  • Subcellular fractions were isolated via sequential centrifugation.
  • Protein identification was performed using two-dimensional (2-D) electrophoresis, proteolytic digestion, and mass spectrometry.

Main Results:

  • Dounce homogenization followed by centrifugation enriched plasma membrane proteins fivefold but contained significant contaminants (22 out of 30 identified proteins).
  • Nitrogen cavitation followed by centrifugation and detergent solubilization (SDS and CHAPS) yielded purer subcellular fractions with less protein overlap.
  • 2-D electrophoresis revealed distinct protein profiles between fractions obtained by the two methods.

Conclusions:

  • Nitrogen cavitation is a more effective method for isolating purer plasma membrane fractions from U937 cells compared to Dounce homogenization.
  • Complete solubilization of membrane proteins is critical for comprehensive proteomic analysis.
  • These findings emphasize the importance of optimized sample preparation for accurate plasma membrane proteomics.

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