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
- Carla Schmidt1,4,7,
- Mads Grønborg1,5,7,
- Jochen Deckert2,6,
- Sergey Bessonov2,
- Thomas Conrad2,
- Reinhard Lührmann2 and
- Henning Urlaub1,3,8
- 1Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany
- 2Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany
- 3Bioanalytics, Department of Clinical Chemistry, University Medical Center Göttingen, 37075 Göttingen, Germany
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↵7 These authors contributed equally to this work.
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
- spliceosome
- quantitative proteomics
- stable isotope labeling with amino acids in cell culture (SILAC)
- isobaric tags for relative and absolute quantification (iTRAQ)
- spectral count
Footnotes
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↵8 Corresponding author
E-mail henning.urlaub{at}mpibpc.mpg.de
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Freely available online through the RNA Open Access option.
- Received July 11, 2013.
- Accepted December 10, 2013.
This article, published in RNA, is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported), as described at http://creativecommons.org/licenses/by-nc/3.0/.