Sizing large proteins and protein complexes by electrospray ionization mass spectrometry and ion mobility

Abstract

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

Elucidating the site of protein-ATP binding by top-down mass spectrometry

“…Because the phase transition from solution to the gas phase is not a subtle effect, it would not be surprising to find some perturbation in the structure of a protein complex upon removal of solvent. In fact, studies suggest that large protein complexes undergo a shrinking process as a result of solvent loss [1]. Therefore, the solution to Breuker and McLafferty's question, "For how long, under what conditions, and to what extent, can solution structure be retained without solvent?"…”

“…With electrospray ionization (ESI) to ionize macromolecules without disrupting covalent bonds while maintaining weak noncovalent interactions, the ESI-MS molecular mass measurement provides direct evidence for protein-ligand associations. The proteinligand interactions are often sufficiently retained upon the transition from solution to the gas phase that the complex size and binding stoichiometry can be measured [1][2][3].Although binding stoichiometry and, in many examples, the relative and absolute solution binding affinities can be measured by ESI-MS methodologies, determining the precise ligand binding site on a target protein is not easily tractable using MS directly. Tandem mass spectrometry (MS/MS) is widely applied to derive the amino acid sequence of small polypeptides (for example, in the so-called bottom-up proteomics approach), and MS/MS of intact proteins (i.e., top-down MS [4]) show potential to be a tool for protein identification and elucidation of post-translational modifications [5].…”

“…With electrospray ionization (ESI) to ionize macromolecules without disrupting covalent bonds while maintaining weak noncovalent interactions, the ESI-MS molecular mass measurement provides direct evidence for protein-ligand associations. The proteinligand interactions are often sufficiently retained upon the transition from solution to the gas phase that the complex size and binding stoichiometry can be measured [1][2][3].…”

Ion mobility–mass spectrometry analysis of large protein complexes

“…Given interpretable MS data, the structural information provided by IM-MS is limited by the IM resolution (R) achieved for the complex of interest. Currently, it is difficult to find examples of IM resolution 410-15 for large protein complexes 13,50 Increasing the maximum IM resolution achievable for proteins and protein complexes is an active area of research. We envision that the information provided by IM-MS could be integrated as restraints, along with other low-resolution structural information, to provide higher-fidelity structure representations of protein assemblies, including those comprised of more than one type of subunit 4,5 .…”

“…A wide range of densities have been used in the literature to describe the packing efficiencies of gas-phase proteins and protein complexes 38,49,50 and the density used here is toward the lower extreme of this range. As such, it is not surprising that some complexes exhibit collision cross-sections that appear substantially more compact than predicted by the general relationship shown in Figure 3b.…”