Mass Spectrometry of Intact Protein Complexes

Protein function is often defined by the interactions proteins form with a range of biomolecules and other proteins. Mass spectrometry (MS), the workhorse of protein identification in the current proteomics effort, is now providing information regarding the noncovalent interaction networks of proteins by introducing intact protein assemblies into the mass spectrometer. This chapter presents examples that highlight the diverse array of complexes amenable to the technique and the insights provided from the data obtained. We have not attempted a comprehensive overview of the field but rather have focused on spectacular advances involving large macromolecular complexes such as ribosomes, proteasomes, viruses, and rotary ATPases. Instrumental considerations required to make these studies are discussed. As well as providing stoichiometric information for these assemblies, MS provides insights into the dynamic regions of these structures that are challenging to other biophysical techniques. Amyloidogenic proteins, which are implicated in a range of degenerative disease states, are also presented. The sensitivity and speed of MS enabling the observation of early transient oligomers of fibril‐forming proteins make the techniques attractive for characterizing oligomerization mechanisms. Small heat shock proteins, which assemble into large polydisperse complexes, can be characterized and the hundreds of coexisting oligomeric states identified, with ion mobility measurements. This combination of approaches enables structural models to be assigned to these species. The functional activity of the HSP90 cycle requires a cooperative effort between various cochaperones and the stoichiometries of the interactions formed during the cycle are difficult to define. MS has not only allowed the real‐time monitoring of complexes formed during this cycle, but also the affinities of each interaction can be quantified. We present recent advances that allow intact membrane protein complexes to be studied by MS, using micelles to protect their transition into the gas phase. Finally, we present the role of MS in integrative approaches to structural biology. Information obtained from MS‐based experiments is now being used as restraints for modeling, essential in the case of complex dynamic assemblies which elude traditional structural biology approaches.