Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method of gas-phase electrophoretic mobility molecular analysis (GEMMA), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions, which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared with other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and X-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm 3 . Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water. (J Am Soc Mass Spectrom 2007, 18, 1206 -1216

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.

Vaults are 13 million Dalton ribonucleoprotein particles with a highly conserved structure. Expression and assembly by multimerization of an estimated 96 copies of a single protein, termed the major vault protein (MVP), is sufficient to form the minimal structure and entire exterior shell of the barrel-shaped vault particle. Multiple copies of two additional proteins, VPARP and TEP1, and a small untranslated vault RNA are also associated with vaults. We used the Sf9 insect cell expression system to form MVP-only recombinant vaults and performed a series of protein-mixing experiments to test whether this particle shell is able to exclude exogenous proteins from interacting with the vault interior. Surprisingly, we found that VPARP and TEP1 are able to incorporate into vaults even after the formation of the MVP vault particle shell is complete. Electrospray molecular mobility analysis and spectroscopic studies of vault-interacting proteins were used to confirm this result. Our results demonstrate that the protein shell of the recombinant vault particle is a dynamic structure and suggest a possible mechanism for in vivo assembly of vault-interacting proteins into preformed vaults. Finally, this study suggests that the vault interior may be functionally interactive with the cellular milieu.Vaults are structurally conserved ribonucleoprotein (RNP) 1 particles that have been implicated in multidrug resistance, nucleocytoplasmic transport, and as scaffolds for both epidermal growth factor signaling and interferon-gamma activated JAK/STAT signaling pathways; however, their precise function remains unclear (1-9). The particles have a capped-barrel morphology with dimensions of approximately 41 × 41 × 72.5 nm (10), and at 13 million Daltons, are the largest known RNP. Vaults have 8-fold symmetry around their longitudinal axis and each half-vault appears identical (8-2-2 symmetry). When plated onto poly-L-lysine coated electron microscopy grids and visualized by freeze-etch platinum shadowing, vaults † This research was supported by grants from the National Science Foundation (MCB-0210690 and CHE-0507929) and the G. Harold and Leila Y. Mathers Charitable Foundation (01124244). The UCLA Functional Proteomics Center was established and equipped by a grant from the W. M. Keck Foundation. JAL acknowledges support from the Jonsson Comprehensive Cancer Center at UCLA, the UCLA-DOE Institute for Genomics and Proteomics, and the National Institutes of Health (RR 20004).*To whom all correspondence should be addressed: Leonard H. Rome, Department of Biological Chemistry, University of California, Los Angeles, 33-131 CHS mail code #173717, 10833 Le Conte Avenue, Los Angeles, California 90095-1737, Tel. 310 825-0709; Fax. 310 206-5272; Email: lrome@mednet.ucla.edu. 1 ABBREVIATIONS: RNP, ribonucleoprotein; MVP, major vault protein; TEP1, telomerase associated protein 1; VPARP, vault poly (ADP-ribose) polymerase; VR, vault RNA; cryoEM, cryoelectron microscopy; TEM, transmission electron microscopy; M-INT, minimal MVP interactio...

Synthesis of well-defined homodimeric protein-polymer conjugates using RAFT polymerization is described.