Pushing the Limits of Ion Mobility Mass Spectrometry to Investigate Large Protein Complexes
Many protein functions depend heavily upon the structure of the proteins and protein complexes. The use of traditional methods (NMR, crystallography, cryo-EM) for protein structure determination is problematic for macromolecular complexes because of signal broadening (NMR) and there are many difficulties obtaining high resolution images (crystallography, cryo-EM), especially for asymmetric assemblies. Recently, mass spectrometry (MS) and ion mobility spectrometry (IMS) studies have suggested that macromolecules in the gas-phase retain elements of solution-phase structure. An emerging area is the study of large biological assemblies (hundred of kDa) in the gas phase. Robinson, Heck, and others have employed mobility separations coupled with MS to characterize large protein complexes, such as the GroEL chaperone complex and the tryptophan RNA binding protein (TRAP) complex. This unique approach does not require crystallization, can determine mass, and is not limited by the heterogeneity of sample. This allows for more biologically relevant measurements, as most cellular conditions are not conducive to crystallization.
One limitation in the study of large protein complexes utilizing IMS-MS is the determination of accurate collision cross sections. We have utilized IMS-MS, using a high resolution home built instrument with a 3 meter drift tube, to determine accurate collision cross sections for a number of protein complexes, including bovine serum albumin, Saccharomyces cerevisiae alcohol dehydrogenase, and rabbit muscle pyruvate kinase. These protein complexes are some of the largest complexes that have been successfully measured with a conventional drift tube to obtain accurate collisional cross sections. This approach provides unique information about the assembly of protein subunits into an active complex, and allows for the study of these complexes under biologically relevant conditions.