Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Summary

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully ¹⁵N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Abstract

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% ¹. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient ². Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.

We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K¹⁵NO₃ as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest ³. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control ⁴.

Introduction

In 1972, Jonathan Singer and Garth Nicolson proposed the fluid mosaic model a structure model of cellular membranes, replacing the protein-lipid-protein sandwich model that was generally accepted in the early 1960s. Singer and Nicolson postulated that the biological membrane can be considered as a two-dimensional liquid where all lipid and protein molecules diffuse freely and easily ⁵. Since that time, structure model of the plasma membrane and knowledge of the membrane composition became even more complex. Particularly, within the plasma membrane, structures such as protein complexes and lipid/sterol based structurally disordered microdomains can be observ....

Protocol

PROCEDURE

Common reagents and buffers used in the extraction protocol:

1. JPL medium for Arabidopsis thaliana suspension cell cultures
   • 3 μM H₃BO₃
   • 3 μM MnSO₄ x H₂O
   • 1.1 μM ZnSO₄ x 7 H₂O
   • 0.15 μM KJ
   • 0.03 μM Na₂MoO₄ x 2 H₂O
   • 3 nM CoCl₂ x 6 H₂O
   • 3 nM CuSO₄ x 5 H₂O
   • 0.9 mM CaCl₂ x 2 H₂O
   • 0.5 mM MgSO₄ x 7 H₂O
   • 0.5 μM FeSO_....

Results

With the presented protocol using metabolically labeled Arabidopsis cell cultures it is possible to isolate plasma membranes from plant tissue (step2, Figures 2 and 4), and enrich for detergent resistant membrane fractions within the plasma membrane (step 3, Figures 3 and 5). Subsequently, the protocol allows extraction of proteins from these detergent resistant membrane fractions (step 4) and digestion of the protein for comparative proteomic analysis (step 5). Finally, the opt.......

Discussion

The protocol presented in this paper contains many steps and all of them are crucial to obtain pure and representative fractionation of the plant plasma membrane into detergent resistant membranes and detergent soluble fractions. Therefore, it is important to follow each step as instructed.

Treatment of the plasma membrane fraction with non-ionic detergent (step 3.2) has the strongest influence on the quality of membrane microdomain fractionation. To obtain reproducible results between differe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
Chemicals were ordered from Sigma-Aldrich unless noted otherwise
Ammonia (stock 25 % solution)	WAKO	010-03166
TiO2 10 μm	GL-Science	5020-75010
Empore Disk C18	Varian	12145004
Polyethylene glycol(PEG 3350)	Sigma	88276
Dextran T500	Roth	9219.2
Trypsin	Promega	V5113
Protease inhibitor cocktail (PIC)	Sigma	P9599
K15NO3	Cambridge Isotope Laboratories	NLM-765-PK
EQUIPMENT
Optima L-80 XP Ultracentrifuge	Beckman
Plate reader	BioTek
EASY-nLC II nano-Liquid Chromatograph	Thermo Scientific
LTQ-Orbitrap mass spectrometer	Thermo Scientific
Centrifuge 5810R	Eppendorf
Centrifuge 5417R	Eppendorf
Thermomixer	Eppendorf
Speed Vac RVC 2-25	Christ
Shaker Unimax 2010	Heidolph