Global Identification of Protein Complexes within the Membrane Proteome of <i>Arabidopsis</i> Roots Using a SEC-MS Approach

Biological processes consist of several consecutive and interacting steps as, for example, in signal transduction cascades or metabolic reaction chains. These processes are regulated by protein–protein interactions and the formation of larger protein complexes, which also occur within biological membranes. To gain a large-scale overview of complex-forming proteins and the composition of such complexes within the cellular membranes of Arabidopsis roots, we use the combination of size-exclusion chromatography and mass spectrometry. First, we identified complex-forming proteins by a retention shift analysis relative to expected retention times of monomeric proteins during size-exclusion chromatography. In a second step we predicted complex composition through pairwise correlation of elution profiles. As result we present an interactome of 963 proteins within cellular membranes of Arabidopsis roots. Identification of complex-forming proteins was highly robust between two independently grown root proteomes. The protein complex composition derived from pairwise correlations of coeluting proteins reproducibly identified stable protein complexes (ribosomes, proteasome, mitochondrial respiratory chain supercomplexes) but showed higher variance between replicates regarding transient interactions (e.g., interactions with kinases) within membrane protein complexes.生物过程由多个连续且相互作用的步骤构成，例如信号转导级联（signal transduction cascade）或代谢反应链。此类过程的调控依赖于蛋白质-蛋白质相互作用以及大型蛋白质复合物的形成，而这类相互作用与复合物形成同样发生在生物膜内部。为了全面解析拟南芥（Arabidopsis）根细胞的生物膜中复合物形成蛋白的分布及其复合物组成，本研究采用尺寸排阻色谱（size-exclusion chromatography）与质谱（mass spectrometry）联用的技术手段。首先，本研究通过对比尺寸排阻色谱中单体蛋白的理论保留时间，基于保留偏移分析鉴定复合物形成蛋白；第二步，通过共洗脱蛋白的洗脱谱两两相关性预测复合物组成。最终，本研究构建了拟南芥根细胞生物膜中包含963种蛋白的相互作用组（interactome）。针对复合物形成蛋白的鉴定在两份独立培养的根蛋白质组样本中具有极高的稳定性。基于共洗脱蛋白两两相关性得到的蛋白质复合物组成，可重复鉴定出核糖体（ribosomes）、蛋白酶体（proteasome）、线粒体呼吸链超复合物（mitochondrial respiratory chain supercomplexes）等稳定蛋白质复合物，但针对膜蛋白复合物内的瞬时相互作用（例如与激酶（kinases）的相互作用），重复样本间的差异相对更高。