Here we describe a detailed protocol for both data collection and interpretation with respect to ion mobility-mass spectrometry analysis of large protein assemblies. Ion mobility is a technique that can separate gaseous ions based on their size and shape. Specifically, within this protocol, we cover general approaches to data interpretation, methods of predicting whether specific model structures for a given protein assembly can be separated by ion mobility, and generalized strategies for data normalization and modeling. The protocol also covers basic instrument settings and best practices for both observation and detection of large noncovalent protein complexes by ion mobility-mass spectrometry. INTRODUCTIONLarge-scale interaction maps suggest a complex interplay of proteins within a myriad of functional assemblies 1,2 . A critical step in assigning functions to these assemblies is to determine their structure 3 . This goal is challenging, as many of these assemblies exist in low-copy numbers within cells, are frequently heterogeneous and may interact only transiently. Consequently, structural information for many protein complexes is not readily accessible by using the classical tools of structural biology (e.g., X-ray crystallography, nuclear magnetic resonance spectroscopy). New approaches are being developed that involve integrating data from a number of lower-resolution experimental methods and by combining distance and interaction restraints from these methods with homology modeling, architectural or even atomic models are being generated 4 . These restraints can be derived from a variety of experimental measurements including MS of intact complexes, chemical cross-linking, fluorescence resonance energy transfer, small angle X-ray scattering, and analytical ultracentrifugation 5,6 . 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 .IM-MS analysis is performed by first ionizing the protein complex of interest. In our experiments, nano-electrospray ionization is used, typically requiring careful preparation procedures for most protein complexes. These procedures, as well as general practical aspects of sample preparation, are detailed in a protocol by Hernández and Robinson 14 . Although they are not discussed in detail here, knowledge of the materials and protocol steps described in that work are critical to the success of the protocol described below.After ionization, ions are injected into a region containing neutral gas at a controlled pressure (e.g., 0.5 mBar of nitrogen gas). Under the influence of a relatively weak electric field, injected ions undergo IM separation [7]...

Auxin is a pivotal plant hormone that controls many aspects of plant growth and development. Perceived by a small family of F-box proteins including transport inhibitor response 1 (TIR1), auxin regulates gene expression by promoting SCF ubiquitin-ligase-catalysed degradation of the Aux/IAA transcription repressors, but how the TIR1 F-box protein senses and becomes activated by auxin remains unclear. Here we present the crystal structures of the Arabidopsis TIR1-ASK1 complex, free and in complexes with three different auxin compounds and an Aux/IAA substrate peptide. These structures show that the leucine-rich repeat domain of TIR1 contains an unexpected inositol hexakisphosphate co-factor and recognizes auxin and the Aux/IAA polypeptide substrate through a single surface pocket. Anchored to the base of the TIR1 pocket, auxin binds to a partially promiscuous site, which can also accommodate various auxin analogues. Docked on top of auxin, the Aux/IAA substrate peptide occupies the rest of the TIR1 pocket and completely encloses the hormone-binding site. By filling in a hydrophobic cavity at the protein interface, auxin enhances the TIR1-substrate interactions by acting as a 'molecular glue'. Our results establish the first structural model of a plant hormone receptor.

Collision cross sections in both helium and nitrogen gases were measured directly using a drift cell with RF ion confinement inserted within a quadrupole/ion mobility/time-of-flight hybrid mass spectrometer (Waters Synapt HDMS, Manchester, U.K.). Collision cross sections for a large set of denatured peptide, denatured protein, native-like protein, and native-like protein complex ions are reported here, forming a database of collision cross sections that spans over 2 orders of magnitude. The average effective density of the native-like ions is 0.6 g cm(-3), which is significantly lower than that for the solvent-excluded regions of proteins and suggests that these ions can retain significant memory of their solution-phase structures rather than collapse to globular structures. Because the measurements are acquired using an instrument that mimics the geometry of the commercial Synapt HDMS instrument, this database enables the determination of highly accurate collision cross sections from traveling-wave ion mobility data through the use of calibration standards with similar masses and mobilities. Errors in traveling-wave collision cross sections determined for native-like protein complexes calibrated using other native-like protein complexes are significantly less than those calibrated using denatured proteins. This database indicates that collision cross sections in both helium and nitrogen gases can be well-correlated for larger biomolecular ions, but non-correlated differences for smaller ions can be more significant. These results enable the generation of more accurate three-dimensional models of protein and other biomolecular complexes using gas-phase structural biology techniques.