The analysis of intact protein assemblies in native-like states by mass spectrometry offers a wealth of information on their biochemical and biophysical properties. Here we show that the Orbitrap mass analyzer can be used to measure protein assemblies of molecular weights approaching one megadalton with sensitivity down to the detection of single ions. Minor instrumental modifications enabled the measurement of various protein assemblies with outstanding mass-spectral resolution.

Gram-negative pathogens commonly use the chaperone-usher pathway to assemble adhesive multisubunit fibers on their surface. In the periplasm, subunits are stabilized by a chaperone that donates a beta strand to complement the subunits' truncated immunoglobulin-like fold. Pilus assembly proceeds through a "donor-strand exchange" (DSE) mechanism whereby this complementary beta strand is replaced by the N-terminal extension (Nte) of an incoming pilus subunit. Using X-ray crystallography and real-time electrospray ionization mass spectrometry (ESI-MS), we demonstrate that DSE requires the formation of a transient ternary complex between the chaperone-subunit complex and the Nte of the next subunit to be assembled. The process is crucially dependent on an initiation site (the P5 pocket) needed to recruit the incoming Nte. The data also suggest a capping reaction displacing DSE toward product formation. These results support a zip-in-zip-out mechanism for DSE and a catalytic role for the usher, the molecular platform at which pili are assembled.

Traditionally, mass spectrometry has been a powerful analytical method enabling the structural analysis of small molecules, and later on peptides and proteins. With the advent of native mass spectrometry, using a combination of electrospray ionisation and time of flight analysis, mass spectrometry could also be applied to the mass determination of large protein complexes such as ribosomes and whole viruses. More recently, ion mobility has been coupled to mass spectrometry providing a new dimension in the analysis of biomolecules, with ion mobility separating ions according to differences in size and shape. In the context of native mass spectrometry, ion mobility mass spectrometry opens up avenues for the detailed structural analysis of large and heterogeneous protein complexes, providing information on the stoichiometry, topology and cross section of these assemblies and their composite subunits. With these characteristics, ion mobility mass spectrometry offers a complementary tool in the context of structural biology. Here, we critically review the development, instrumentation, approaches and applications of ion mobility in combination with mass spectrometry, focusing on the analysis of larger proteins and protein assemblies (185 references).

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