Abstract
Most catalytic, structural and regulatory functions of the cell are carried out by functional modules, typically complexes containing or consisting of proteins. The composition and abundance of these complexes and the quantitative distribution of specific proteins across different modules is therefore of major significance in basic and translational biology. To date, the systematic detection and quantification of protein complexes has remained technically challenging. The chromatographic separation of native protein complexes followed by the mass spectrometric analysis of the proteins contained in sequential fractions results in potentially thousands of protein elution profiles from which, in principle, the presence of specific complexes can be inferred. However, the de novo inference of protein complexes from such datasets has so far remained limited with regard to selectivity and the retrieval of quantitative information.We recently developed a variant of this strategy, complex-centric proteome profiling, which extends the concepts of targeted proteomics to the level of native protein complex analysis. The complex-centric workflow consists of size exclusion chromatography (SEC) to fractionate native protein complexes, DIA/SWATH mass spectrometry to precisely quantify the proteins in each SEC fraction based on a consistent set of peptides, and targeted, complex-centric analysis where prior information from generic protein interaction maps is used to detect and quantify protein complexes with high selectivity and statistical error control via the computational framework CCprofiler. Complex-centric proteome profiling captures the majority of proteins in complex-assembled state and reveals their organization into hundreds of complexes and complex variants observable in a given cellular state. The protocol is applicable to genetically unaltered tissue cultures and adaptable to primary tissue. At present it requires approximately 8 days of wet-lab work, 15 days of MS measurement time and 7 days of computational analysis.
First Lanthanide Complex for De Novo Phasing in Native Protein Crystallography at 1 Å Radiation
