Mass spectrometry-based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

Abstract

The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. Knowing the proteins-and their respective quantities-at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing. Here, we applied three independent mass spectrometry (MS)-based approaches for quantification of these proteins: (1) metabolic labeling by SILAC, (2) chemical labeling by iTRAQ, and (3) label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes. In total we were able to quantify 157 proteins by at least two of the three approaches. Our quantification shows that only a very small subset of spliceosomal proteins (the U5 and U2 Sm proteins, a subset of U5 snRNP-specific proteins, and the U2 snRNP-specific proteins U2A' and U2B'') remains unaltered upon transition from the B to the C complex. The MS-based quantification approaches classify the majority of proteins as dynamically associated specifically with the B or the C complex. In terms of experimental procedure and the methodical aspect of this work, we show that metabolically labeled spliceosomes are functionally active in terms of their assembly and splicing kinetics and can be utilized for quantitative studies. Moreover, we obtain consistent quantification results from all three methods, including the relatively straightforward and inexpensive label-free spectral count technique.

Keywords: isobaric tags for relative and absolute quantification (iTRAQ); quantitative proteomics; spectral count; spliceosome; stable isotope labeling with amino acids in cell culture (SILAC).

FIGURE 1.

Purification of the spliceosomal B and C complexes for proteomic analysis and iTRAQ and SILAC quantification. (A) B and C complexes were purified from “heavy” or “light” SILAC NEs, respectively. The complexes were allowed to assemble onto MS2-tagged pre-mRNA for 6 or 180 min, respectively. The complexes were then isolated by gradient centrifugation and affinity purification; isolated complexes were pooled in equal amounts; the proteins were separated by gel electrophoresis; and the peptides generated were analyzed by LC-MS/MS. (B) B and C complexes were purified from “normal” (light) NE. The proteins were separated by gel electrophoresis, and after in-gel digestion, the peptides generated were analyzed by LC-MS/MS for proteomic analysis, or the peptides generated from the B complex were labeled with iTRAQ reagent 115 and those generated from the C complex were labeled with iTRAQ reagent 116. After pooling, the samples were analyzed by LC-MS/MS (iTRAQ quantification).

FIGURE 2.

Metabolically labeled NEs retained full catalytic activity, as shown by analyzing the B and C complexes purified from “heavy” or “light” SILAC NEs, respectively. (A) The splicing kinetics were determined from aliquots of splicing reactions taken from 0–180 min and analyzed by denaturing gel electrophoresis. Pre-mRNA and splicing products were visualized by autoradiography. Splicing products first appeared after 10 min. (B) The spliceosomal complex formation was assayed by native agarose gel electrophoresis and visualized by autoradiography. The A and B complexes were first observed after 2 and 4 min, respectively, while the C complex first appeared after 10–15 min. (C) The RNA compositions of purified B (“light” SILAC NE) and C (“heavy” SILAC NEs) complexes were analyzed by denaturing gel electrophoresis and visualized by silver staining (lanes 1,3) or autoradiography (lanes 2,4). B complexes contained U1, U2, U4, U5, and U6 snRNA (lane 1) and a large amount of pre-mRNA (lane 2). C complexes contained U2, U5, and U6 snRNA (lane 3) and splicing products and reduced amounts of pre-mRNA (lane 4).

FIGURE 3.

Relative protein abundances of the Sm proteins and the U2 and U5 snRNP-specific proteins obtained by spectral count, SILAC, and iTRAQ. The B:C ratios are shown for the Sm proteins (A), the U2 snRNP-specific proteins (B), and the U5 snRNP specific proteins (C). Ratios of proteins in B versus C complex are plotted on a logarithmic scale; error bars, SD between the two biological replicates. “1” indicates that a protein is present in these complexes in a 1:1 ratio. The different shading of the bars represents the ratios of proteins in B and C complexes derived from the values obtained by SILAC, iTRAQ, and spectral count, respectively (see Table 1; Supplemental Tables).

FIGURE 4.

Relative protein abundances in the B and C complexes for the U1, U4/U6, and U4/U6.U5 snRNP-specific proteins and the LSm proteins, as obtained by SILAC, iTRAQ, and spectral count. (A) B:C ratios for U1 snRNP-specific proteins. No protein ratios were obtained for U1-C from the spectral count since this protein was completely absent from the C complex. (B) B:C ratios for the U4/U6- and U4/U6.U5-specific proteins. (C) B:C ratios for the LSm proteins. No protein ratio was obtained from the spectral count for LSm4 since this protein was absent from the C complex. LSm5 was not identified or quantified. For details, see legend to Figure 3.

FIGURE 5.

Relative protein abundances of the B- and C- specific proteins. (A) B:C ratios for B-specific proteins. (B) B:C ratios for C-specific proteins. For several proteins, no values for spectral count were obtained as these proteins had spectra only in the B (hSnu23, UBL5, Npw38, SPF30) or the C (PPIL3b, FRG1, NOSIP, C1orf55, FAM50B, C9orf78, DGCR14, CCDC130, NKAP, ZCCHC10, CDK10, TTC14, DDX34, PRKRIP1) complex. For details, see legend to Figure 3.

FIGURE 6.

Relative protein abundances of the step 2 factors, the EJC and the hPrp19/CDC5L complex–specific proteins obtained by SILAC, iTRAQ, and spectral count. (A) B:C ratios for step 2 factors and the EJC. No protein ratio was obtained from the spectral count for hPrp18 since this protein was absent from the B complex. (B) B:C ratios for the hPrp19/CDC5L complex–specific proteins. For CTNNBL1, no clear association to either the B or the C complex could be determined. For details, see legend to Figure 3.