A Proteomic Strategy to Discover the Composition, Localization, and Dynamics of Endogenous Protein Complexes in Arabidopsis Leaves

A given eukaryotic cell contains thousands of protein complexes that enable  the cell to generate and respond to signals, fine tune and integrate metabolic activities, and carry out complicated mechanical tasks. Therefore broad knowledge about protein complex composition and dynamics is needed to analyze systems-level behaviors.  To date, affinity purification-mass spectrometry and yeast-two-hybrid are the most widely used methods for high throughput analysis of protein complexes. However, these methods are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. To overcome these problems, we recently developed a new method that combines size exclusion chromatography with quantitative MS to analyze thousands of proteins leading to the discovery of hundreds of novel protein complexes  (Aryal et al., 2014).  We have recently expanded this technique in several important ways. First we have incorporated stable isotope labeling to anlayze how protein complexes rearrange in respose to metabolic stress. Second, we developed a new method to predict protein complex composition by combining SEC with an orthogonal ion exchange chromatography separation to generate abundance profiles of thousands of proteins using MS1 extracted ion chromatograms. These abundance profiles were subjected to clustering analysis to identify proteins that co-fractionate, and are putative subunits of native complexes. Last, our analysis has been extended to include membrane-associated proteins. Our update will include validations using both analyses of known protein complexes and Arabidopsis mutant in predicted complex subunits in order to identify true positives and false negatives.