AUTHOR=Nguyen Truong Van , Gupta Ravi , Annas Dicky , Yoon Jinmi , Kim Yu-Jin , Lee Gi Hyun , Jang Jeong Woo , Park Kang Hyun , Rakwal Randeep , Jung Ki-Hong , Min Cheol Woo , Kim Sun Tae TITLE=An Integrated Approach for the Efficient Extraction and Solubilization of Rice Microsomal Membrane Proteins for High-Throughput Proteomics JOURNAL=Frontiers in Plant Science VOLUME=12 YEAR=2021 URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.723369 DOI=10.3389/fpls.2021.723369 ISSN=1664-462X 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.