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Lipids are highly structurally diverse molecules involved in a wide variety of biological processes. Here, we use high precision ion mobility-mass spectrometry to compile a structural database of 456 mass-resolved collision cross sections (CCS) of sphingolipid and glycerophospholipid species. Our CCS database comprises sphingomyelin, cerebroside, ceramide, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidic acid classes. Primary differences observed are between lipid categories, with sphingolipids exhibiting 2-6% larger CCSs than glycerophospholipids of similar mass, likely a result of the sphingosine backbone's restriction of the sn1 tail length, limiting gas-phase packing efficiency. Acyl tail length and degree of unsaturation are found to be the primary structural descriptors determining CCS magnitude, with degree of unsaturation being four times as influential per mass unit. The empirical CCS values and previously unmapped quantitative structural trends detailed in this work are expected to facilitate prediction of CCS in broadscale lipidomics research.
The inward rectifier potassium (Kir) channel Kir4.1 () carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)--(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC value of 0.97 M and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC = 9 M) at -120 mV. In thallium (Tl) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction () in rat plasma ( = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
Translocator Protein (18 kDa, TSPO) is regarded as a useful biomarker for neuroinflammation imaging. TSPO PET imaging could be used to understand the role of neuroinflammation in brain diseases and as a tool for evaluating novel therapeutic effects. As a promising TSPO probe, [F]DPA-714 is highly specific and offers reliable quantification of TSPO in vivo. In this study, we further radiosynthesized and evaluated another novel TSPO probe, 2-(7-butyl-2-(4-(2-[F]fluoroethoxy)phenyl)-5-methylpyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide ([F]VUIIS1018A), which features a 700-fold higher binding affinity for TSPO than that of [F]DPA-714. We evaluated the performance of [F]VUIIS1018A using dynamic in vivo PET imaging, radiometabolite analysis, in vitro autoradiography assays, biodistribution analysis, and blocking assays. In vivo study using this probe demonstrated high signal-to-noise ratio, binding potential (BP), and binding specificity in preclinical neuroinflammation studies. Taken together, these findings indicate that [F]VUIIS1018A may serve as a novel TSPO PET probe for neuroinflammation imaging.
Copyright © 2018. Published by Elsevier Masson SAS.
BACKGROUND - The human body contains numerous long-lived proteins which deteriorate with age, typically by racemisation, deamidation, crosslinking and truncation. Previously we elucidated one reaction responsible for age-related crosslinking, the spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine and cysteine. This resulted in non-disulphide covalent crosslinks. The current paper outlines a novel posttranslational modification (PTM) in human proteins, which involves the addition of dehydroalanylglycine (DHAGly) to Lys residues.
METHODS - Human lens digests were examined by mass spectrometry for the presence of (DHA)Gly (+144.0535 Da) adducts to Lys residues. Peptide model studies were undertaken to elucidate the mechanism of formation.
RESULTS - In the lens, this PTM was detected at 18 lysine sites in 7 proteins. Using model peptides, a pathway for its formation was found to involve initial formation of the glutathione degradation product, γ-Glu(DHA)Gly from oxidised glutathione (GSSG). Once the Lys adduct formed, the Glu residue was lost in a hydrolytic mechanism apparently catalysed by the ε-amino group of the Lys.
CONCLUSIONS - This discovery suggests that within cells, the functional groups of amino acids in proteins may be susceptible to modification by reactive metabolites derived from GSSG.
GENERAL SIGNIFICANCE - Our finding demonstrates a novel +144.0535 Da PTM arising from the breakdown of oxidised glutathione.
Copyright © 2018. Published by Elsevier B.V.
Secondary metabolite discovery requires an unbiased, comprehensive workflow to detect unknown unknowns for which little to no molecular knowledge exists. Untargeted mass spectrometry-based metabolomics is a powerful platform, particularly when coupled with ion mobility for high-throughput gas-phase separations to increase peak capacity and obtain gas-phase structural information. Ion mobility data are described by the amount of time an ion spends in the drift cell, which is directly related to an ion's collision cross section (CCS). The CCS parameter describes the size, shape, and charge of a molecule and can be used to characterize unknown metabolomic species. Here, we describe current and emerging applications of ion mobility-mass spectrometry for prioritization, discovery and structure elucidation, and spatial/temporal characterization.
Copyright © 2017 Elsevier Ltd. All rights reserved.
Characterization of methylenedianiline (MDA) 2-ring isomers (2,2'-, 2,4'-, and 4,4'-MDA) is reported using matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), a common technique used for characterizing synthetic polymers. MDA is a precursor to methylene diphenyl diisocyanate (MDI), a hard block component in polyurethane (PUR) synthesis. This work focuses on comparing MALDI results to those of our previous electrospray ionization-mass spectrometry (ESI-MS) studies. In ESI, 2-ring MDA isomers formed single unique [M + H] (199 Da) parent ions, whereas in MALDI each isomer shows significant formation of three precursor ions: [M - H] = 197 Da, [M] = 198 Da, and [M + H] = 199 Da. Structures and schemes are proposed for the MALDI fragment ions associated with each precursor ion. Ion mobility-mass spectrometry (IM-MS), tandem mass spectrometry (MS/MS), and computational methods were all critical in determining the structures for both precursor and fragment ions as well as the fragmentation mechanisms. The present study indicates that the [M - H] and [M] ions are formed by the MALDI process, explaining why they were not observed with ESI.
Collision cross section (CCS) measurements resulting from ion mobility-mass spectrometry (IM-MS) experiments provide a promising orthogonal dimension of structural information in MS-based analytical separations. As with any molecular identifier, interlaboratory standardization must precede broad range integration into analytical workflows. In this study, we present a reference drift tube ion mobility mass spectrometer (DTIM-MS) where improvements on the measurement accuracy of experimental parameters influencing IM separations provide standardized drift tube, nitrogen CCS values (CCS) for over 120 unique ion species with the lowest measurement uncertainty to date. The reproducibility of these CCS values are evaluated across three additional laboratories on a commercially available DTIM-MS instrument. The traditional stepped field CCS method performs with a relative standard deviation (RSD) of 0.29% for all ion species across the three additional laboratories. The calibrated single field CCS method, which is compatible with a wide range of chromatographic inlet systems, performs with an average, absolute bias of 0.54% to the standardized stepped field CCS values on the reference system. The low RSD and biases observed in this interlaboratory study illustrate the potential of DTIM-MS for providing a molecular identifier for a broad range of discovery based analyses.
Yatakemycin (YTM) is an extraordinarily toxic DNA alkylating agent with potent antimicrobial and antitumor properties and is the most recent addition to the CC-1065 and duocarmycin family of natural products. Though bulky DNA lesions the size of those produced by YTM are normally removed from the genome by the nucleotide-excision repair (NER) pathway, YTM adducts are also a substrate for the bacterial DNA glycosylases AlkD and YtkR2, unexpectedly implicating base-excision repair (BER) in their elimination. The reason for the extreme toxicity of these lesions and the molecular basis for the way they are eliminated by BER have been unclear. Here, we describe the structural and biochemical properties of YTM adducts that are responsible for their toxicity, and define the mechanism by which they are excised by AlkD. These findings delineate an alternative strategy for repair of bulky DNA damage and establish the cellular utility of this pathway relative to that of NER.
Recently, zebrafish and human cytochrome P450 (P450) 27C1 enzymes have been shown to be retinoid 3,4-desaturases. The enzyme is unusual among mammalian P450s in that the predominant oxidation is a desaturation and in that hydroxylation represents only a minor pathway. We show by proteomic analysis that P450 27C1 is localized to human skin, with two proteins of different sizes present, one being a cleavage product of the full-length form. P450 27C1 oxidized all--retinol to 3,4-dehydroretinol, 4-hydroxy (OH) retinol, and 3-OH retinol in a 100:3:2 ratio. Neither 3-OH nor 4-OH retinol was an intermediate in desaturation. No kinetic burst was observed in the steady state; neither the rate of substrate binding nor product release was rate-limiting. Ferric P450 27C1 reduction by adrenodoxin was 3-fold faster in the presence of the substrate and was ∼5-fold faster than the overall turnover. Kinetic isotope effects of 1.5-2.3 (on / ) were observed with 3,3-, 4,4-, and 3,3,4,4-deuterated retinol. Deuteration at C-4 produced a 4-fold increase in 3-hydroxylation due to metabolic switching, with no observable effect on 4-hydroxylation. Deuteration at C-3 produced a strong kinetic isotope effect for 3-hydroxylation but not 4-hydroxylation. Analysis of the products of deuterated retinol showed a lack of scrambling of a putative allylic radical at C-3 and C-4. We conclude that the most likely catalytic mechanism begins with abstraction of a hydrogen atom from C-4 (or possibly C-3) initiating the desaturation pathway, followed by a sequential abstraction of a hydrogen atom or proton-coupled electron transfer. Adrenodoxin reduction and hydrogen abstraction both contribute to rate limitation.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
A chemical structure (CS) identifies the connectivities between atoms, and the nature of those connections, for a given elemental composition. For chiral molecules, in addition to the identification of CS, the identification of the correct absolute configuration (AC) is also needed. Several chiral natural products are known whose CSs were initially misidentified and later corrected, and these errors were often discovered during the total synthesis of natural products. In this work, we present a new and convenient approach that can be used with Raman optical activity (ROA) and vibrational circular dichroism (VCD) spectroscopies, to distinguish between the correct and incorrect CSs of chiral compounds. This approach involves analyzing the spectral similarity overlap between experimental spectra and those predicted with advanced quantum chemical theories. Significant labor needed for establishing the correct CSs via chemical syntheses of chiral natural products can thus be avoided.
© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.