Large-scale proteomic analysis of the human spliceosome

Abstract

In a previous proteomic study of the human spliceosome, we identified 42 spliceosome-associated factors, including 19 novel ones. Using enhanced mass spectrometric tools and improved databases, we now report identification of 311 proteins that copurify with splicing complexes assembled on two separate pre-mRNAs. All known essential human splicing factors were found, and 96 novel proteins were identified, of which 55 contain domains directly linking them to functions in splicing/RNA processing. We also detected 20 proteins related to transcription, which indicates a direct connection between this process and splicing. This investigation provides the most detailed inventory of human spliceosome-associated factors to date, and the data indicate a number of interesting links coordinating splicing with other steps in the gene expression pathway.

Figure 1

On-line LC MS/MS mass spectrometric analysis of the spliceosome. (A1) Sum of the ion intensity measured by mass spectrometry at any point in the chromatogram for the mass range m/z = 350–550 (total ion chromatogram[TIC]), (B1) m/z = 550–750, and (C1) m/z = 750–1400. (Insets) Current for an ion of specific mass (m/z window 0.2) eluting around the marked time in the chromatogram. (A2,B2,C2) Mass spectra obtained at the marked time in the corresponding upper panels. Several peptides coelute. (Insets) Zoom of the peak marked with an asterisk. (A3,B3,C3) Fragmentation spectra of the peak marked with an asterisk in the corresponding mass spectra above. The insets show that there was high resolution and the ability to determine the charge state by the differences between isotopic peaks. Open circles mark the peaks corresponding to predicted fragments for the peptide sequence retrieved by the database search. The insets show that there was high-quality data for the fragments, which aided the database search and validation. (D) The proteins identified by the peptides shown in A–C are also independently identified by a large number of other peptide fragmentation spectra, leading to substantial sequence coverage of 32%–57% for these three proteins.

Figure 2

Protein abundance index (PAI) for the identified proteins. Plot of PAI index, which is defined as the number of identified peptides divided by the number of calculated, observable peptides, plotted for the identified proteins in seven different classes. The individual spots represent the PAI for each protein in the category. The bar shaded in gray extents to the average value of the PAI for each category. The white bar encompasses 95% of the proteins in each category.

Figure 3

Classification of the identified proteins into eight functional classes.

Figure 4

Virtual 2D gel of proteins identified in the spliceosome preparation. The coordinates are the theoretical molecular mass and isoelectric point for each protein. The gray circles represent factors identified in this study, and the black circles represent proteins identified in this and our previous study (Neubauer et al. 1998). The box indicates the coordinate space spanned by our previous investigation using 2D gel electrophoresis.