An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics.

Truong Van Nguyen,Gi Hyun Lee,Jinmi Yoon,Jeong Woo Jang,Yu-Jin Kim,Ravi Gupta,Cheol Woo Min,Kang Hyun Park,Ki-Hong Jung,Dicky Annas,Sun Tae Kim,Randeep Rakwal

doi:10.3389/fpls.2021.723369

Abstract

The preparation of microsomal membrane proteins (MPs) is critically important to microsomal proteomics. To date most research studies have utilized an ultracentrifugation-based approach for the isolation and solubilization of plant MPs. However, these approaches are labor-intensive, time-consuming, and unaffordable in certain cases. Furthermore, the use of sodium dodecyl sulfate (SDS) and its removal prior to a mass spectrometry (MS) analysis through multiple washing steps result in the loss of proteins. To address these limitations, this study introduced a simple micro-centrifugation-based MP extraction (MME) method from rice leaves, with the efficacy of this approach being compared with a commercially available plasma membrane extraction kit (PME). Moreover, this study assessed the subsequent solubilization of isolated MPs in an MS-compatible surfactant, namely, 4-hexylphenylazosulfonate (Azo) and SDS using a label-free proteomic approach. The results validated the effectiveness of the MME method, specifically in the enrichment of plasma membrane proteins as compared with the PME method. Furthermore, the findings showed that Azo demonstrated several advantages over SDS in solubilizing the MPs, which was reflected through a label-free quantitative proteome analysis. Altogether, this study provided a relatively simple and rapid workflow for the efficient extraction of MPs with an Azo-integrated MME approach for bottom-up proteomics.

Highlights

Microsomes are cell membrane-derived vesicles that are formed during the lysis of plant tissues (LaMontagne et al, 2016)
To compare the protein profiles of the isolated membrane proteins (MPs), a labelfree quantitative proteomic approach was utilized, which led to the identification of 18,240 peptides and 15,737 unique peptides that matched with 4,045 protein groups, resulting in an average sequence coverage of 13% (Figure 1A)
The three replicates of the same sample in plasma membrane extraction kit (PME) showed less than 12.5% of the coefficient of variation (CV) values, while less than 4.2% of the CV values were observed in the case of the microcentrifugation-based MP extraction (MME) method (Figure 1A)

Summary

Introduction

Microsomes are cell membrane-derived vesicles that are formed during the lysis of plant tissues (LaMontagne et al, 2016). The largescale isolation of MPs for proteome analysis has primarily relied on ultracentrifugation-based methods due to multiple advantages such as the higher purity of products, suitability in a large volume of samples, and accurate separation of subcellular compartments based on own sedimentation rates (Yang D. et al, 2020) These ultracentrifugation-based methods require expensive instrumentation, skilled technicians, and a large amount of starting material, which limits their large-scale utilization (Wang et al, 2015; Alqurashi et al, 2017; Meisrimler et al, 2017; Yang D. et al, 2020). The development of relatively simpler methods for the isolation of MPs is necessary and is a prerequisite for microsomal proteomic studies

Methods

Results

Conclusion