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.
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.
Carla
C
Schmidt
Mads
M
Grønborg
Jochen
J
Deckert
Sergey
S
Bessonov
Thomas
T
Conrad
Reinhard
R
Lührmann
Henning
H
Urlaub
D006801:Humans; D007553:Isotope Labeling; D011506:Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing / Q000235:genetics; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D011506:Proteins; D020543:Proteome; D012322:RNA Precursors
isobaric tags for relative and absolute quantification (iTRAQ); quantitative proteomics; spectral count; spliceosome; stable isotope labeling with amino acids in cell culture (SILAC)