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  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.

Schmidt, C., Groenborg, M., Deckert, J., Bessonov, S., Conrad, T., Lührmann, R., et al. (2014). 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. RNA, 20(3), 406-420. doi:10.1261/rna.041244.113.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0018-65D7-2 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0027-CD73-9
Genre: Journal Article

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Schmidt, C.1, Author              
Groenborg, M., Author
Deckert, J.2, Author              
Bessonov, S.2, Author              
Conrad, T.2, Author              
Lührmann, R.2, Author              
Urlaub, H.1, Author              
Affiliations:
1Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society, ou_578613              
2Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society, ou_578576              

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 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.

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Language(s): eng - English
 Dates: 2014-01-212014-03
 Publication Status: Published in print
 Pages: 15
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 Table of Contents: -
 Rev. Method: Peer
 Identifiers: DOI: 10.1261/rna.041244.113
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Title: RNA
Source Genre: Journal
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Pages: - Volume / Issue: 20 (3) Sequence Number: - Start / End Page: 406 - 420 Identifier: -