Adventures in Molecular Wonderland: Exploring the Complexity of Protein Complexes Through Complexome Profiling - And the Role of Size-Exclusion Chromatography

Abstract

Proteins do not act on their own but form different complexes in the cell to exert their biological function. This dynamic process of protein complex assembly is fine-tuned, in part by post-translational modifications (PTMs), like phosphorylation. Elucidating the different complexes of a cell, the complexome, and studying these upon perturbations, allows us to understand cellular functions and how they are interrupted in certain conditions or diseases, in a systems-wide view. Complexome profiling poses several analytical challenges, for which sophisticated methods have been developed to purify and analyze these protein complex mixtures. These methods lean heavily on Mass Spectrometry (MS), as it can handle complex samples, with relatively high sensitivity and in a high-throughput manner. The protein complexes are obtained by targeted approaches, like IP-MS, AP-MS, and Proximity Labelling, or by untargeted approaches, based on the biochemical separation of the complexes, like Size-Exclusion Chromatography (SEC). SEC purifies complexes from native conditions, in a high-throughput manner, with a smaller risk of interfering with protein function compared to targeted approaches. As complexome profiling produces a lot of data, several tools exist to analyze this systematically, additionally providing validation tools to minimize the risk of false positives. This review presents some recent literature on SEC-based complexome profiling, which data-analysis toolkits have been developed, how assembly- or condition-specific PTMs could be studied, which caveats these approaches still possess, and which further improvements are being made or what should be an area of interest for follow-up studies.