System-wide profiling of protein-complexes via size exclusion chromatography– mass spectrometry (SEC-MS)

In living cells most proteins are organized in stable or transient functional assemblies known as protein complexes, which control a multitude of vital cellular processes such as cell cycle progression, metabolism, and signal transduction. System-wide workflows for analysis of protein complexes using mass spectrometry based proteomics consists of a biochemical fractionation methods which take advantage of biophysical features such as charge or mass of protein complexes. In this project, we apply size exclusion chromatography to investigate protein assemblies from a Jurkat whole cell lysate. We introduced a fast and robust in-plate sample preparation workflow which allows for replicates to be performed with fewer technical hurdles and with a high degree of reproducibility) We acquired the data in SWATH-mode in combination with a high-throughput LC-system (EVOSEP one). The bioinformatics pipeline builds on the OpenSWATH workflow combined with CCprofiler, a tool for statistical scoring of protein co-elution profiles. This method enabled us to identify more than 100 protein complexes from mammalian cells within one day of measurement.