Characterization of the human COP9 signalosome complex using affinity purification and mass spectrometry.
The COP9 signalosome (CSN) is a multiprotein complex that plays a critical role in diverse cellular and developmental processes in various eukaryotic organisms. Despite of its significance, current understanding of the biological functions and regulatory mechanisms of the CSN complex is still very limited. To unravel these molecular mechanisms, we have performed a comprehensive proteomic analysis of the human CSN complex using a new purification method and quantitative mass spectrometry. Purification of the human CSN complex from a stable 293 cell line expressing N-terminal HBTH-tagged CSN5 subunit was achieved by high-affinity streptavidin binding with TEV cleavage elution. Mass spectrometric analysis of the purified CSN complex has revealed the identity of its composition as well as N-terminal modification and phosphorylation of the CSN subunits. N-terminal modifications were determined for seven subunits, six of which have not been reported previously, and six novel phosphorylation sites were also identified. Additionally, we have applied the newly developed MAP-SILAC and PAM-SILAC methods to decipher the dynamics of the human CSN interacting proteins. A total of 52 putative human CSN interacting proteins were identified, most of which are reported for the first time. In comparison to PAM-SILAC results, 20 proteins were classified as stable interactors, whereas 20 proteins were identified as dynamic ones. This work presents the first comprehensive characterization of the human CSN complex by mass spectrometry-based proteomic approach, providing valuable information for further understanding of CSN complex structure and biological functions.
Fang
Lei
L
Departments of Physiology & Biophysics and Developmental & Cell Biology, University of California, Irvine, California 92697, USA.
Wang
Xiaorong
X
Yamoah
Kosj
K
Chen
Phang-lang
PL
Pan
Zhen-Qiang
ZQ
Huang
Lan
L
D000075686:COP9 Signalosome Complex; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D002853:Chromatography, Liquid; D006801:Humans; D007553:Isotope Labeling; D007668:Kidney / Q000166:cytology; D013058:Mass Spectrometry / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics
D046912:Multiprotein Complexes; D021122:Protein Subunits; D020543:Proteome; D010447:Peptide Hydrolases; D000075686:COP9 Signalosome Complex