Analyzing the protein assembly and dynamics of the human spliceosome with SILAC

Quantitative mass spectrometry has become an indispensable tool in proteomic studies. Numerous methods are available and can be applied to approach different issues. In most studies these issues include the quantitative comparison of different cell states, the identification of specific interaction partners or determining degrees of posttranslational modification. In this chapter we describe a SILAC-based quantification in order to analyze dynamic protein changes during the assembly of the human spliceosome on a pre-mRNA in vitro. We provide protocols for assembly of spliceosomes on pre-mRNA (including generation of pre-mRNAs and preparation of nuclear extracts), quantitative mass spectrometry (SILAC labeling, sample preparation), and data analysis to generate timelines for the dynamic protein assembly.