A Conformation-Specific Approach to Native Top-down Mass Spectrometry.

Native top-down mass spectrometry is a powerful approach for characterizing proteoforms and has recently been applied to provide similarly powerful insights into protein conformation. Current approaches, however, are limited such that structural insights can only be obtained for the entire conformational landscape in bulk or without any direct conformational measurement. We report a new ion-mobility-enabled method for performing native top-down MS in a conformation-specific manner.

Enhanced Declustering Enables Native Top-Down Analysis of Membrane Protein Complexes using Ion-Mobility Time-Aligned Fragmentation.

Native mass spectrometry (MS) is proving to be a disruptive technique for studying the interactions of proteins, necessary for understanding the functional roles of these biomolecules. Recent research is expanding the application of native MS towards membrane proteins directly from isolated membrane preparations or from purified detergent micelles. The former results in complex spectra comprising several heterogeneous protein complexes; the latter enables therapeutic protein targets to be screened against multiplexed preparations of compound libraries.

Understanding the structural dynamics of human islet amyloid polypeptide: Advancements in and applications of ion-mobility mass spectrometry.

Human islet amyloid polypeptide (hIAPP) forms amyloid deposits that contribute to β-cell death in pancreatic islets and are considered a hallmark of Type II diabetes Mellitus (T2DM). Evidence suggests that the early oligomers of hIAPP formed during the aggregation process are the primary pathological agent in islet amyloid induced β-cell death. The self-assembly mechanism of hIAPP, however, remains elusive, largely due to limitations in conventional biophysical techniques for probing the distribution or capturing detailed structures of the early, structurally dynamic oligomers.

Linking Gas-Phase and Solution-Phase Protein Unfolding via Mobile Proton Simulations.

Native mass spectrometry coupled to ion mobility (IM-MS) combined with collisional activation (CA) of ions in the gas phase () is an important method for the study of protein unfolding. It has advantages over classical biophysical and structural techniques as it can be used to analyze small volumes of low-concentration heterogeneous mixtures while maintaining solution-like behavior and does not require labeling with fluorescent or other probes. It is unclear, however, whether the unfolding observed during collision activation experiments mirrors solution-phase unfolding.

Cyclic Ion Mobility-Collision Activation Experiments Elucidate Protein Behavior in the Gas Phase.

Ion mobility coupled to mass spectrometry (IM-MS) is widely used to study protein dynamics and structure in the gas phase. Increasing the energy with which the protein ions are introduced to the IM cell can induce them to unfold, providing information on the comparative energetics of unfolding between different proteoforms. Recently, a high-resolution cyclic IM-mass spectrometer (cIM-MS) was introduced, allowing multiple, consecutive tandem IM experiments (IM) to be carried out.

Analysis of Proline Substitutions Reveals the Plasticity and Sequence Sensitivity of Human IAPP Amyloidogenicity and Toxicity.

Pancreatic amyloid formation by the polypeptide IAPP contributes to β-cell dysfunction in type 2 diabetes. There is a 1:1 correspondence between the ability of IAPP from different species to form amyloid and the susceptibility of the organism to develop diabetes. Rat IAPP is non-amyloidogenic and differs from human IAPP at six positions, including three proline replacements: A25P, S28P, and S29P.