Absolute and relative quantification of proteins in large protein-RNA assemblies by mass spectrometry

Abstract

The majority of cellular processes is driven by protein complexes. These emerge as multimeric protein assemblies or are complexed with ligands such as RNA or DNA. To understand the details of the cellular process, an analysis of the protein complexes is required. This involves the identification of the protein components, the determination of the protein stoichiometries, the relative quantification of different complex states, and the study of protein-protein or protein-ligand interactions. Several mass spectrometry-based techniques have been developed to tackle these problems and greatly facilitating the high throughput analysis of protein complexes. In this study, these methods have been applied to the spliceosome, which is a protein-RNA machinery that catalyzes the excision of introns and the ligation of exons during eukaryotic pre-mRNA splicing. We used absolute and relative quantification by mass spectrometry to characterize different spliceosomal complexes or subcomplexes, in terms of their protein composition and protein stoichiometry. One spliceosomal subcomplex, the hPrp19/CDC5L complex, consists of seven individual proteins (hPrp19, Hsp70, CTNNBL1, PRL1, CDC5L, AD-002, and SPF27) and plays a crucial role in the assembly of a catalytically active spliceosome. The exact protein stoichiometries within this particular protein complex have not yet been investigated. We therefore set up a mass spectrometry-based quantification method and used the hPrp19/CDC5L complex to implement the methodology of absolute quantification (AQUA) with the help of synthetic standard peptides in combination with multiple reaction monitoring (MRM). The analyses revealed that the hPrp19/CDC5L complex consists of four copies of hPrp19, two copies of CDC5L, and one copy each of SPF27, PRL1, and CTNNBL1. In a different series of investigation, we set out to make a high throughput relative quantification of two distinct spliceosomal complexes, namely the pre-catalytic and the catalytically active spliceosomes (spliceosomal B and C complexes). For this purpose, a relative quantification approach involving iTRAQ (isobaric tags for relative and absolute quantification) labeling of in-gel digested proteins was optimized and applied to the two complexes. The results were compared to relative quantification by metabolic labeling (SILAC; stable isotope labeling with amino acids in cell culture) and semi-quantitative spectral count from proteomic analysis of the B and C complexes. We found an overall good agreement for the used quantification methods. Several proteins were found to be pre-dominantly associated with spliceosomal B or C complexes, and only few proteins were found to be present in equal amounts within the two complexes. The high dynamics of the spliceosome during its assembly pathway was thus clearly demonstrated. In a final scene of investigation, we analyzed the dynamic protein changes during the pre-mRNA splicing process. To this end, the protein compositions on different pre-mRNAs at different time points were compared by stable isotope labeling with amino acids in cell culture (SILAC). The generated timelines for the assembly of whole groups of spliceosomal proteins during spliceosomal assembly and splicing catalysis are extremely helpful in understanding the dynamic process of pre-mRNA splicing.