Simple Strategy of Integrating the Micro-centrifugation-based Extraction and 4-Hexylphenylazosulfonate for the Preparation of Plant Microsomal Proteins for Bottom-Up Proteomics

ABSTRACT: The preparation of high-quality microsomal proteins (MPs) is of critical importance to high-throughput microsomal proteomics. The isolation of MPs for proteomic analysis has to date relied on Ultracentrifugation-based methods (UBMs), however, these methods are labor-intensive, time-consuming, and unaffordable to many laboratories. Here, we report a simple method developed by integrating the micro-centrifugation-based extraction (MME) method and 4-hexylphenylazosulfonate (Azo) surfactant for the isolation and solubilization of MPs from rice leaves. A mass spectrometry-based (MS) label-free proteomic approach was used for a comprehensive quantitative comparison of the extractability between the MME method and the plasma membrane extraction (PME) kit and of the ability to solubilize proteins between Azo and sodium dodecyl sulfate (SDS). Results showed that the MME method can not only effectively isolate MPs from rice leaves for a bottom-up proteomic analysis but also performs better than the PME kit in the enrichment of plasma membrane (PM) proteins. Additionally, Azo was found to effectively solubilize total proteins, including the MPs from rice leaves for bottom-up proteomics with performance comparable to that of SDS. Azo can be integrated with the MME approach as a simple and effective strategy to prepare MPs and could replace SDS for high-throughput proteomic studies. This is the first study revealing the applicability of the micro-centrifugation-based method and Azo in plant proteomics

INSTRUMENT(S): Q Exactive

ORGANISM(S): Oryza Sativa Subsp. Japonica (rice)

TISSUE(S): Leaf

SUBMITTER: Sun Tae Kim  

LAB HEAD: Sun Tae Kim

PROVIDER: PXD025132 | Pride | 2021-09-29

REPOSITORIES: Pride