Systematic characterization of nuclear proteome during apoptosis: a quantitative proteomic study by differential extraction and stable isotope labeling.
Identification and characterization of the nuclear proteome is important for detailed understanding of multiple signaling events in eukaryotic cells. Toward this goal, we extensively characterized the nuclear proteome of human T leukemia cells by sequential extraction of nuclear proteins with different physicochemical properties using three buffer conditions. This large scale proteomic study also tested the feasibility and technical challenges associated with stable isotope labeling by amino acids in cell culture (SILAC) to uncover quantitative changes during apoptosis. Analyzing proteins from three nuclear fractions extracted from naive and apoptotic cells generated 780,530 MS/MS spectra that were used for database searching using the SEQUEST algorithm. This analysis resulted in the identification and quantification of 1,174 putative nuclear proteins. A number of known nuclear proteins involved in apoptosis as well as novel proteins not known to be part of the nuclear apoptotic machinery were identified and quantified. Consistent with SILAC-based quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear invaginations during apoptosis.
Department of Cell Biology, Center for Vascular Biology, Center for Cell Analysis and Modeling, University of Connecticut School of Medicine, Farmington, Connecticut 06030, USA.
Sun-Il
SI
Hwang
Deborah H
DH
Lundgren
Viveka
V
Mayya
Karim
K
Rezaul
Ann E
AE
Cowan
Jimmy K
JK
Eng
David K
DK
Han
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry / Q000648:ultrastructure; D015536:Down-Regulation; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D019169:Jurkat Cells; D013058:Mass Spectrometry; D008928:Mitochondria / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D012984:Software; D015854:Up-Regulation
D009687:Nuclear Proteins; D020543:Proteome