Electrospray ionization mass spectrometry and ion mobility analysis of the 20S proteasome complex

Abstract

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.

Electrospray–differential mobility analysis of bionanoparticles

“…This work was followed over the next decade by several reports from Allmaier et al [11], Wick et al [12] and de la Mora et al [13] related to characterization of several other proteins, viruses and polymers. Starting in 2006 as seen in Figures 1.3A and 1.3B a dramatic increase in the number of publications and citations related to ES-DMA occurred with contributions from Biswas et al [14], Zachariah et al [15], Loo et al [16], Hogan et al [17], Pergantis et al [18], Pease et al [19] and Hackley et al [20]. Although developed primarily for size determination, with an increasing pool of users, several orthogonal utilities have been found for this technique a detailed discussion of which will be appear later in Chapter 3.…”

Ion mobility–mass spectrometry analysis of large protein complexes

“…One very recent addition to this series of biophysical tools is ion mobility separation coupled to mass spectrometry (IM-MS). IM is an established technique for studying shape and conformation in small molecules and individual proteins in the gas phase 7-10 but has only recently been applied to intact protein complexes 11,12 . When IM is coupled with MS, mass and consequently subunit composition can be determined simultaneously with the overall topology of protein complexes 10,12,13 .…”

Gas-phase H/D exchange and collision cross sections of hemoglobin monomers, dimers, and tetramers

“…In the absence of water, monomeric proteins can adopt compact conformations similar to the solution conformations, but also extended conformations far removed from the native state [2,3]. Much of the current understanding of the "size" of protein ions in the gas phase comes from physical methods of measuring collision cross sections using ion mobility spectrometry at pressures of a few Torr [4 -7], at atmospheric pressure [8], or at pressures of ca. 1 ϫ 10 Ϫ3 Torr [7,9,10].…”

“…Ion mobility measurements of complex macromolecular protein complexes at atmospheric pressure [8] or at a pressures of ca. 1 ϫ 10 Ϫ3 Torr have shown some success [9,10].…”