Evaluation of Protein Stoichiometry within Protein-RNA Complexes by Multiple Reaction Monitoring (MRM) in Comparison to MS Standard Methods

摘要

Many cellular processes are driven by protein complexes. Although identification of the protein components in such complexes has become routine, determination of their protein stoichiometry still represents a formidable challenge. One way to tackle this challenge is absolute quantification (AQUA) using synthetic standard peptides (AQUA peptides, [1]). We use Multiple Reaction Monitoring (MRM, [2]) in combination with Absolute Quantification (AQUA) to determine the protein stoichiometry within spliceosomal complexes and demonstrate the utility of the method in the analysis of the native spliceosomal hPrp19/CDC5 complex. The spliceosome is a multi-megadalton machinery that catalyzes the excision of introns and the ligation of exons during eukaryotic pre-mRNA splicing. It assembles from different uridine-rich snRNPs (small nuclear ribonucleo-protein particles) and numerous non-snRNP proteins. During splicing it passes through different functional states that differ in their RNA and protein composition. The hPrp19/CDC5 complex [3] plays a crucial role in the assembly of a fully catalytically active spliceosome, presumably by stabilizing the RNA interaction network in the catalytical core. It consists of seven proteins and represents an ideal test system.