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

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).