Ion mobility mass spectrometry (IM-MS) allows separation of native protein ions into “conformational families”. Increasing the IM resolving power should allow finer structural information to be obtained and can be achieved by increasing the length of the IM separator. This, however, increases the time that protein ions spend in the gas phase and previous experiments have shown that the initial conformations of small proteins can be lost within tens of milliseconds. Here, we report on investigations of protein ion stability using a multipass traveling wave (TW) cyclic IM (cIM) device. Using this device, minimal structural changes were observed for Cytochrome C after hundreds of milliseconds, while no changes were observed for a larger multimeric complex (Concanavalin A). The geometry of the instrument (Q-cIM-ToF) also enables complex tandem IM experiments to be performed, which were used to obtain more detailed collision-induced unfolding pathways for Cytochrome C. The instrument geometry provides unique capabilities with the potential to expand the field of protein analysis via IM-MS.

Our study aimed to establish the complete structure of the main dihydroxy conjugated triene issued from the lipoxygenation (soybean enzyme) of docosahexaenoic acid, named PDX, an isomer of protectin/neuroprotectin D1 (PD1/NPD1) described by Bazan and Serhan. NMR approaches and other chemical characterization (e.g. GC-MS, HPLC and LC-MS/MS) indicated that PDX is 10(S),17(S)-dihydroxy-docosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid. The use of (18)O(2) and mass spectrometry showed that PDX is a double lipoxygenation product. Its structure differs from PD1, with E,Z,E geometry (PDX) instead of E,E,Z (PD1) and S configuration at carbon 10 instead of R. PDX inhibits human blood platelet aggregation at sub-micromolar concentrations.

Ozone-induced dissociation (OzID) is a novel ion activation technology that exploits the gas-phase reaction between mass-selected ions and ozone inside a mass spectrometer to assign sites of unsaturation in complex lipids. Since it was first demonstrated [ Thomas et al. Anal. Chem. 2008 , 80 , 303 ], the method has been widely deployed for targeted lipid structure elucidation but its application to high throughput and liquid chromatography-based workflows has been limited due to the relatively slow nature of the requisite ion-molecule reactions that result in long ion-trapping times and consequently low instrument duty cycle. Here, the implementation of OzID in a high-pressure region, the ion-mobility spectrometry cell, of a contemporary quadrupole time-of-flight mass spectrometer is described. In this configuration, a high number density of ozone was achieved and thus abundant and diagnostic OzID product ions could be observed even on the timescale of transmission through the reaction region (ca. 20-200 ms), representing a 50-1000-fold improvement in performance over prior OzID implementations. Collisional activation applied prereaction was found to yield complementary and structurally informative product ions arising from ozone- and collision-induced dissociation. Ultimately, the compatibility of this implementation with contemporary ultrahigh performance liquid chromatography is demonstrated with the resulting hyphenated approach showing the ability to separate and uniquely identify isomeric phosphatidylcholines that differ only in their position(s) of unsaturation.

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 n ) to be carried out. We describe a tandem IM technique for defining detailed protein unfolding pathways and the dynamics of disordered proteins. The method involves multiple rounds of IM separation and collision activation (CA): IM-CA-IM and CA-IM-CA-IM. Here, we explore its application to studies of a model protein, cytochrome C, and dimeric human islet amyloid polypeptide (hIAPP), a cytotoxic and amyloidogenic peptide involved in type II diabetes. In agreement with prior work using single stage IM-MS, several unfolding events are observed for cytochrome C. IM n -MS experiments also show evidence of interconversion between compact and extended structures. IM n -MS data for hIAPP shows interconversion prior to dissociation, suggesting that the certain conformations have low energy barriers between them and transition between compact and extended forms.

Gas‐phase protein separation by ion mobility: With its ability to separate the Parkinson's disease protein α‐synuclein and its autoproteolytic products—despite the small concentrations of the latter—ion‐mobility MS has enabled the characterization of intermediate fragments in in vitro oligomerization‐aggregation. In particular, a possible key fragment, the highly aggregating C‐terminal fragment, αSyn(72–140), has been revealed.