The ability to preserve noncovalent, macromolecular assemblies intact in the gas phase has paved the way for mass spectrometry to characterize ions of increasing size and become a powerful tool in the field of structural biology. Tandem mass spectrometry experiments have the potential to expand the capabilities of this technique through the gas-phase dissociation of macromolecular complexes, but collisions with small gas atoms currently provide very limited fragmentation. One alternative for dissociating large ions is to collide them into a surface, a more massive target. Here, we demonstrate the ability and benefit of fragmenting large protein complexes and inorganic salt clusters by surface-induced dissociation (SID), which provides more extensive fragmentation of these systems and shows promise as an activation method for ions of increasing size. ver the past two to three decades, mass spectrometry (MS) has expanded significantly, from its early use as a technique for measuring the isotopes of elements and analyzing volatile compounds, to a technique that is now routinely used to study nonvolatile molecules and large macromolecular complexes. Increasingly, mass spectrometry and ion mobility/mass spectrometry are described as structural biology tools. Mass spectrometry has recently provided insights on posttranslational modifications [1], mono-and polydisperse subunit stoichiometry [2], subunit organization [3,4], and noncovalent protein-ligand binding sites [5]. In addition to revealing structural information, mass spectrometry can be used to monitor dynamic processes, such as protein complex assembly [6], protein-substrate and protein-protein interactions [7,8], and substratespecific conformational changes [9].One of the limitations of current technology, however, is the fact that commercial instrumentation is still hampered by the amount of dissociation that can be induced from large biomolecular complexes. Often, MS has to be combined with many solution-based experiments (H/D exchange plus digestion, chemical crosslinking plus digestion, limited proteolysis, solution disruption by changes in ionic strength) because the MS instruments commercially available do not provide extensive dissociation of these massive complexes. A typical dissociation result that is achieved is ejection of a monomer subunit as illustrated here (Figure 1) for a small heat shock protein (sHSP) dodecamer, consisting of 12 subunits each weighing 16.9 kDa [8,10,11]. The 16.9 kDa monomer typically carries away a large percentage of the charge of the original complex with the remainder of the charge on the 11-mer that weighs 186 kDa. Investigators have concluded that this behavior, which has been observed by several research groups for many different noncovalent protein complexes, happens because the low-energy collisions with a gas initiate unfolding of one of the protein subunits [12][13][14]. As this subunit unfolds, it gains surface area, thus allowing it to accept more charge. Eventually, when the unfolding protein and the remainde...

As scientists begin to appreciate the extent to which quaternary structure facilitates protein function, determination of the subunit arrangement within non-covalent protein complexes is increasingly important. While native mass spectrometry shows promise for the study of non-covalent complexes, few developments have been made towards the determination of subunit architecture, and no mass spectrometry activation method yields complete topology information. Here we illustrate the activation and dissociation by surface-induced dissociation of a heterohexamer, toyocamycin nitrile hydratase, directly into its constituent trimers. We propose that the single-step nature of this activation in combination with high energy deposition allows for dissociation prior to significant unfolding or other large-scale rearrangement. This method can potentially allow for dissociation of a protein complex into subcomplexes facilitating the mapping of subunit contacts and thus determination of quaternary structure of protein complexes.

Background: Toxin-antitoxin (TA) systems are broadly conserved in the bacterial kingdom. Results: Brucella abortus RNase toxin, BrnT, has a RelE-like fold and is neutralized by its antitoxin, BrnA. brnTA transcription is activated by a number of environmental stressors. Conclusion: BrnTA is a novel, stress-regulated TA system. Significance: This study structurally and functionally defines a novel member of the RelE toxin family.

Characterization of endogenous metabolites and xenobiotics is essential to deconvoluting the genetic and environmental causes of disease. However, surveillance of chemical exposure and disease-related changes in large cohorts requires an analytical platform that offers rapid measurement, high sensitivity, efficient separation, broad dynamic range, and application to an expansive chemical space. Here, we present a novel platform for small molecule analyses that addresses these requirements by combining solid-phase extraction with ion mobility spectrometry and mass spectrometry (SPE-IMS-MS). This platform is capable of performing both targeted and global measurements of endogenous metabolites and xenobiotics in human biofluids with high reproducibility (CV 6 3%), sensitivity (LODs in the pM range in biofluids) and throughput (10-s sample-to-sample duty cycle). We report application of this platform to the analysis of human urine from patients with and without type 1 diabetes, where we observed statistically significant variations in the concentration of disaccharides and previously unreported chemical isomers. This SPE-IMS-MS platform overcomes many of the current challenges of large-scale metabolomic and exposomic analyses and offers a viable option for population and patient cohort screening in an effort to gain insights into disease processes and human environmental chemical exposure.

This paper describes the development of a molecularly imprinted polymer (MIP) for theophylline that can be used for electrochemical sensing. Theophylline is a commonly used medication for the treatment of asthma. Due to its very narrow therapeutic index, it may have toxic and potentially fatal effects on the individual. Electrochemical detection of theophylline is difficult, because its molecular structure and standard reduction potential are very similar to that of caffeine. A new method for fabricating molecularly imprinted polymers is proposed utilizing methylene green. Poly(methylene green)(PMG), prepared by electropolymerization of an azine, methylene green, was imprinted for theophylline. PMG-based MIP-coated electrodes showed sensitivity towards the presence of the imprint molecule in solutions, as well as selectivity for the imprint over the interferent molecule caffeine. The PMG-based MIP-coated electrode described in this paper had an improved selectivity factor and reproducibility compared to other theophylline-imprinted MIP-coated electrodes in literature.