SPE-IMS-MS: An automated platform for sub-sixty second surveillance of endogenous metabolites and xenobiotics in biofluids.

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.

An unusual dimeric small heat shock protein provides insight into the mechanism of this class of chaperones.

Small heat shock proteins (sHSPs) are virtually ubiquitous stress proteins that are also found in many normal tissues and accumulate in diseases of protein folding. They generally act as ATP-independent chaperones to bind and stabilize denaturing proteins that can be later reactivated by ATP-dependent Hsp70/DnaK, but the mechanism of substrate capture by sHSPs remains poorly understood. A majority of sHSPs form large oligomers, a property that has been linked to their effective chaperone action. We describe AtHsp18.5 from Arabidopsis thaliana, demonstrating that it is dimeric and exhibits robust chaperone activity, which adds support to the model that suboligomeric sHSP forms are a substrate binding species. Notably, like oligomeric sHSPs, when bound to substrate, AtHsp18.5 assembles into large complexes, indicating that reformation of sHSP oligomeric contacts is not required for assembly of sHSP-substrate complexes. Monomers of AtHsp18.5 freely exchange between dimers but fail to coassemble in vitro with dodecameric plant cytosolic sHSPs, suggesting that AtHsp18.5 does not interact by coassembly with these other sHSPs in vivo. Data from controlled proteolysis and hydrogen-deuterium exchange coupled with mass spectrometry show that the N- and C-termini of AtHsp18.5 are highly accessible and lack stable secondary structure, most likely a requirement for substrate interaction. Chaperone activity of a series of AtHsp18.5 truncation mutants confirms that the N-terminal arm is required for substrate protection and that different substrates interact differently with the N-terminal arm. In total, these data imply that the core α-crystallin domain of the sHSPs is a platform for flexible arms that capture substrates to maintain their solubility.

Molecular structure and function of the novel BrnT/BrnA toxin-antitoxin system of Brucella abortus.

Type II toxin-antitoxin (TA) systems are expressed from two-gene operons that encode a cytoplasmic protein toxin and its cognate protein antitoxin. These gene cassettes are often present in multiple copies on bacterial chromosomes, where they have been reported to regulate stress adaptation and persistence during antimicrobial treatment. We have identified a novel type II TA cassette in the intracellular pathogen Brucella abortus that consists of the toxin gene, brnT, and its antitoxin, brnA. BrnT is coexpressed and forms a 2:2 tetrameric complex with BrnA, which neutralizes BrnT toxicity. The BrnT(2)-BrnA(2) tetramer binds its own promoter via BrnA, and autorepresses its expression; its transcription is strongly induced in B. abortus by various stressors encountered by the bacterial cell during infection of a mammalian host. Although highly divergent at the primary sequence level, an atomic resolution (1.1 Å) crystal structure of BrnT reveals a secondary topology related to the RelE family of type II ribonuclease toxins. However, overall tertiary structural homology to other RelE family toxins is low. A functional characterization of BrnT by site-directed mutagenesis demonstrates a correspondence between its in vitro activity as a ribonuclease and control of bacteriostasis in vivo. We further present an analysis of the conserved and variable features of structure required for RNA scission in BrnT and the RelE toxin family. This structural investigation informs a model of the RelE-fold as an evolutionarily flexible scaffold that has been selected to bind structurally disparate antitoxins, and exhibit distinct toxin activities including RNA scission and DNA gyrase inhibition.

Determinants of gas-phase disassembly behavior in homodimeric protein complexes with related yet divergent structures.

The overall structure of a protein-protein complex reflects an intricate arrangement of noncovalent interactions. Whereas intramolecular interactions confer secondary and tertiary structure to individual subunits, intermolecular interactions lead to quaternary structure--the ordered aggregation of separate polypeptide chains into multisubunit assemblies. The specific ensemble of noncovalent contacts dictates the stability of subunit folds, enforces protein-protein binding specificity, and determines multimer stability. Consequently, noncovalent architecture is likely to play a role in the gas-phase dissociation of these assemblies during tandem mass spectrometry (MS/MS). To further advance the applicability of MS/MS to analytical problems in structural biology, a better understanding of the interplay between the structures and fragmentation behaviors of noncovalent protein complexes is essential. The present work constitutes a systematic study of model protein homodimers (bacteriophage N15 Cro, bacteriophage λ Cro, and bacteriophage P22 Arc) with related but divergent structures, both in terms of subunit folds and protein-protein interfaces. Because each of these dimers has a well-characterized structure (solution and/or crystal structure), specific noncovalent features could be correlated with gas-phase disassembly patterns as studied by collision-induced dissociation, surface-induced dissociation, and ion mobility. Of the several respects in which the dimers differed in structure, the presence or absence of intermolecular electrostatic contacts exerted the most significant influence on the gas-phase dissociation behavior. This is attributed to the well-known enhancement of ionic interactions in the absence of bulk solvent. Because salt bridges are general contributors to both intermolecular and intramolecular stability in protein complexes, these observations are broadly applicable to aid in the interpretation or prediction of dissociation spectra for noncovalent protein assemblies.

Revealing the quaternary structure of a heterogeneous noncovalent protein complex through surface-induced dissociation.

As scientists begin to appreciate the extent to which quaternary structure facilitates protein function, determination of the subunit arrangement within noncovalent protein complexes is increasingly important. While native mass spectrometry shows promise for the study of noncovalent complexes, few developments have been made toward the determination of subunit architecture, and no mass spectrometry activation method yields complete topology information. Here, we illustrate the 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.

Comparison of electropolymerized thiazine dyes as an electrocatalyst in enzymatic biofuel cells and self powered sensors.

This paper details the comparison of different electropolymerized thiazine electrocatalysts for NADH oxidation. Electropolymerized thiazines have been shown to be electrocatalysts for NADH, but no comprehensive comparison of their properties in the same environment has been performed. The electropolymerization and electrocatalysis is very dependent on chemical and electrochemical environment, so the thiazines (methylene green, methylene blue, toluidine blue, azure a, azure b, and azure c) were all electropolymerized in the same chemical and electrochemical environment and tested for NADH electrocatalysis. All of the thiazines can be electropolymerized to form stable polymer modified electrodes on glassy carbon electrodes and all shown electrocatalytic activity toward NADH. However, each polymer has different properties and therefore would be employed in different applications, depending on whether open circuit potential, current density, or lifetime is the most important condition of the biofuel cell. This paper further compares NAD-dependent glucose dehydrogenase bioelectrocatalysis with poly(methylene green) and poly(methylene blue) electrocatalysts in terms of sensitivity to glucose and biofuel cell performance.

Surface-induced dissociation shows potential to be more informative than collision-induced dissociation for structural studies of large systems.

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.

Poly(methylene green) employed as molecularly imprinted polymer matrix for electrochemical sensing.

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.