Mass spectrometric characterization of the affinity-purified human 26S proteasome complex.
The 26S proteasome is a multisubunit complex responsible for degradation of ubiquitinated substrates, which plays a critical role in regulating various biological processes. To fully understand the function and regulation of the proteasome complex, an important step is to elucidate its subunit composition and posttranslational modifications. Toward this goal, a new affinity purification strategy has been developed using a derivative of the HB tag for rapid isolation of the human 26S proteasome complex for subsequent proteomic analysis. The purification of the complex is achieved from stable 293 cell lines expressing a HB-tagged proteasome subunit and by high-affinity streptavidin binding with TEV cleavage elution. The complete composition of the 26S proteasome complex, including recently assigned new subunits, is identified by LC-MS/MS. In addition, all known proteasome activator proteins and components involved in the ubiquitin-proteasome degradation pathway are identified. Aside from the subunit composition, the N-terminal modification and phosphorylation of the proteasome subunits have been characterized. Twelve novel phosphorylation sites from eight subunits have been identified, and N-terminal modifications are determined for 25 subunits, 12 of which have not been previously reported in mammals. We also observe different N-terminal processing of subunit Rpn2, which results in identification of two different N-termini of the protein. This work presents the first comprehensive characterization of the human 26S proteasome complex by affinity purification and tandem mass spectrometry. The detailed proteomic profiling obtained here is significant to future studies aiming at a complete understanding of the structure-function relationship of the human 26S proteasome complex.
Wang
Xiaorong
X
Department of Physiology and Biophysics, University of California, Irvine, California 92697-4560, USA.
Chen
Chi-Fen
CF
Baker
Peter R
PR
Chen
Phang-lang
PL
Kaiser
Peter
P
Huang
Lan
L
D051046:Amino-Acid N-Acetyltransferase; D002460:Cell Line; D002846:Chromatography, Affinity; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D010446:Peptide Fragments; D010748:Phosphopeptides; D051046:Amino-Acid N-Acetyltransferase; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease