Expression profiling of lymphocyte plasma membrane proteins.

Area of Science:

• Proteomics
• Cell Biology
• Biochemistry

Background:

• Mammalian plasma membrane proteins possess physicochemical properties that hinder analysis via traditional two-dimensional electrophoresis.
• A robust method is needed to systematically identify and quantify these proteins.

Purpose of the Study:

• To develop and validate a novel strategy for the comprehensive analysis of plasma membrane proteins.
• To identify plasma membrane proteins in murine T cell hybridoma and splenocytes.
• To investigate differential protein expression in response to specific stimuli.

Main Methods:

• In vivo cell surface labeling using a water-soluble biotinylation reagent.
• Cell lysis, membrane purification, and affinity capture of biotinylated proteins.
• Separation by solution-phase isoelectric focusing and SDS-PAGE, followed by identification using LC-ESI/MS/MS.

Main Results:

• Identification of 42 plasma membrane proteins from a murine T cell hybridoma and 46 from primary murine splenocytes.
• Discovery of unexpected proteins including nicastrin, osteoclast inhibitory lectin, and a hypothetical transmembrane protein.
• Observed differential expression of CD69, MHC class II, GITR, and surface IgD/M in stimulated splenocytes, confirmed by Western blot and flow cytometry.

Conclusions:

• The developed approach provides a powerful and versatile strategy for investigating plasma membrane proteomes.
• This method enables the study of differential surface protein expression across various cell types and conditions.
• The findings offer new insights into cell surface protein dynamics and potential biomarkers.