Optimization of a detergent-based protocol for membrane proteins purification from mammalian cells

Riccardo Rampado, Federica Giordano, Laura Moracci, Sara Crotti, Paolo Caliceti, Marco Agostini, Francesca Taraballi

Research output: Contribution to journalArticlepeer-review

Abstract

Membrane proteins constitute around 20–30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of “omics” techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers.

Original languageEnglish (US)
Article number114926
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume219
DOIs
StatePublished - Sep 20 2022

Keywords

  • Detergent
  • Extraction
  • Mammalian cells
  • Membrane proteins
  • Protocol

ASJC Scopus subject areas

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry

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