Electrospray ionization mass spectrometry and ion mobility analysis of the 20S proteasome complex.
Mass spectrometry and gas phase ion mobility [gas phase electrophoretic macromolecule analyzer (GEMMA)] with electrospray ionization were used to characterize the structure of the noncovalent 28-subunit 20S proteasome from Methanosarcina thermophila and rabbit. ESI-MS measurements with a quadrupole time-of-flight analyzer of the 192 kDa alpha7-ring and the intact 690 kDa alpha7beta7beta7alpha7 are consistent with their expected stoichiometries. Collisionally activated dissociation of the 20S gas phase complex yields loss of individual alpha-subunits only, and it is generally consistent with the known alpha7beta7beta7alpha7 architecture. The analysis of the binding of a reversible inhibitor to the 20S proteasome shows the expected stoichiometry of one inhibitor for each beta-subunit. Ion mobility measurements of the alpha7-ring and the alpha7beta7beta7alpha7 complex yield electrophoretic diameters of 10.9 and 15.1 nm, respectively; these dimensions are similar to those measured by crystallographic methods. Sequestration of multiple apo-myoglobin substrates by a lactacystin-inhibited 20S proteasome is demonstrated by GEMMA experiments. This study suggests that many elements of the gas phase structure of large protein complexes are preserved upon desolvation, and that methods such as mass spectrometry and ion mobility analysis can reveal structural details of the solution protein complex.
Loo
Joseph A
JA
Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California 90095-1570, USA. JLoo@chem.ucla.edu
Berhane
Beniam
B
Kaddis
Catherine S
CS
Wooding
Kerry M
KM
Xie
Yongming
Y
Kaufman
Stanley L
SL
Chernushevich
Igor V
IV
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry; D006736:Horses; D017020:Methanosarcina / Q000201:enzymology; D036103:Nanotechnology; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D061988:Proteasome Inhibitors; D011817:Rabbits; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D015853:Cysteine Proteinase Inhibitors; D061988:Proteasome Inhibitors; C067713:lactacystin; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine