Chemoproteomic profiling of kinases in live cells using electrophilic sulfonyl triazole probes

Sulfonyl-triazoles are a new class of electrophiles that mediate covalent reaction with tyrosine residues on proteins through sulfur-triazole exchange (SuTEx) chemistry. Recent studies demonstrate the broad utility and tunability of SuTEx chemistry for chemical proteomics and protein ligand discovery. Here, we present a strategy for mapping protein interaction networks of structurally complex binding elements using functionalized SuTEx probes. We show that the triazole leaving group (LG) can serve as a releasable linker for embedding hydrophobic fragments to direct molecular recognition while permitting efficient proteome-wide identification of binding sites in live cells. We synthesized a series of SuTEx probes functionalized with a lipid kinase fragment binder for discovery of ligandable tyrosines residing in catalytic and regulatory domains of protein and metabolic kinases in live cells. We performed competition studies with kinase inhibitors and substrates to demonstrate that probe binding is occurring in an activity-dependent manner. Our functional studies led to discovery of probe-modified sites within the C2 domain that were important for downregulation of protein kinase C-alpha in response to phorbol ester activation. Our proof of concept studies highlight the triazole LG of SuTEx probes as a traceless linker for locating protein binding sites targeted by complex recognition elements in live cells.

Sulfonyl-triazole probes modified with a kinase recognition element are developed for live cell activity-based profiling to identify tyrosine sites located in catalytic and regulatory domains that are important for kinase function.     

Bronsted acid-promoted olefin aziridination and formal anti-aminohydroxylation

A straightforward synthesis of aziridines is reported from an electron-rich azide (alkyl or aryl azide), electron-deficient olefin, and triflic acid in cold acetonitrile. The only coproduct of the reaction is dinitrogen (N2). Active ester substrates bearing a nucleophilic carbonyl engage the putative protonated aziridine intermediate to produce the product of olefin aminohydroxylation in which the nitrogen is benzyl protected and the oxygen is acylated. The possibility that a triazoline need not be an intermediate in aziridine production is advanced.     

Octylgermane on gold: synthesis, oxidation, and pattern formation

Chemisorbed monolayers of octylgermane (C8H17GeH3) on gold have been formed by vapor deposition in ultrahigh vacuum. The monolayer has been characterized by X-ray photoelectron and reflection absorption infrared spectroscopies (XPS and RAIRS) and scanning tunneling microscopy (STM). XPS data indicate the monolayer can be oxidized by exposure to ozone. STM images exhibit a complex pattern which can be modeled as strain-mediated spinodal decomposition.     

Covalent binding of human serum albumin and ovalbumin by chloramine-T and chemical modification of the proteins

The covalent binding of ³⁵S-chloramine-T to human resum albumin (HSA) and ovalbumin is described. At pH 6.5, up to 24 chloramine-T molecules were found to be covalently bound per molecule of HSA; with ovalbumin the binding was only 5–7 molecule per protein molecule. Binding was accompanied by extensive modification of methionine, cysteine, histidine, tyrosine and lysine. Three new peaks appeared in the amino acid profiles of the modified proteins; two were identified as 1-aminoadipic acid (oxidation of lysine) and 3-chlorotyrosine. The most sites for covalent binding are lysine residues.     

A multiresidue pesticide monitoring procedure using gas chromatography/mass spectrometry and selected ion monitoring for the determination of pesticides containing nitrogen, sulfur, and/or oxygen in fruits and vegetables

A procedure for the analysis of over 100 pesticides that only contain combinations of carbon, hydrogen, nitrogen, sulfur, and oxygen (i.e., no phosphorous or halogen atoms) has been developed. The procedure employs gas chromatography with a mass selective detector (GC/MSD), electron impact ionization, and selected ion monitoring. This GC/MSD procedure provided lower limits of quantitation and increased confirmational data compared to the traditional element-selective GC procedures that are commonly used for the detection of this "class" of pesticides. These analytical improvements were demonstrated by 26 pesticides that were detected at ng/g levels in a variety of fruit and vegetable matrixes using this procedure; these pesticides were missed by the traditional element-selective GC procedures. Validation of the procedure was performed using acetone extraction with solid-phase extraction cleanup. Twenty representative target pesticides were used to demonstrate repeatability and linearity of the chromatographic response and recovery from fruit and vegetable matrixes.     

Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins fromE. coli

The high-level expression of recombinant gene products in the gramnegative bacteriumEscherichia coli often results in the misfolding of the protein of interest and its subsequent degradation by cellular proteases or its deposition into biologically inactive aggregates known as inclusion bodies. It has recently become clear that in vivo protein folding is an energy-dependent process mediated by two classes of folding modulators. Molecular chaperones, such as the DnaK-DnaJ-GrpE and GroEL-GroES systems, suppress off-pathway aggregation reactions and facilitate proper folding through ATP-coordinated cycles of binding and release of folding intermediates. On the other hand, folding catalysts (foldases) accelerate rate-limiting steps along the protein folding pathway such as thecis/trans isomerization of peptidyl-prolyl bonds and the formation and reshuffling of disulfide bridges. Manipulating the cytoplasmic folding environment by increasing the intracellular concentration of all or specific folding modulators, or by inactivating genes encoding these proteins, holds great promise in facilitating the production and purification of heterologous proteins. Purified folding modulators and artificial systems that mimic their mode of action have also proven useful in improving the in vitro refolding yields of chemically denatured polypeptides. This review examines the usefulness and limitations of molecular chaperones and folding catalysts in both in vivo and in vitro folding processes.     

Pseudooctahedral complexes of vanadium(III): electronic structure investigation by magnetic and electronic spectroscopy

A variety of physical methods has been used to probe the non-Kramers, S = 1, V(III) ion in two types of pseudooctahedral complexes: V(acac)(3), where acac = anion of 2,4-pentanedione, and VX(3)(thf)(3), where thf = tetrahydrofuran and X = Cl and Br. These methods include tunable frequency and high-field electron paramagnetic resonance (HFEPR) spectroscopy (using frequencies of approximately 95-700 GHz and fields up to 25 T) in conjunction with electronic absorption, magnetic circular dichroism (MCD), and variable-temperature variable-field MCD (VTVH-MCD) spectroscopies. Variable-temperature magnetic susceptibility and field-dependent magnetization measurements were also performed. All measurements were conducted on complexes in the solid state (powder or mull samples). The field versus sub-THz wave quantum energy dependence of observed HFEPR resonances yielded the following spin Hamiltonian parameters for V(acac)(3): D = +7.470(1) cm(-1); E = +1.916(1) cm(-1); g(x) = 1.833(4); g(y) = 1.72(2); g(z) = 2.03(2). For VCl(3)(thf)(3), HFEPR detected a single zero-field transition at 15.8 cm(-1) (474 GHz), which was insufficient to determine the complete set of spin Hamiltonian parameters. For VBr(3)(thf)(3), however, a particularly rich data set was obtained using tunable-frequency HFEPR, and analysis of this data set gave the folowing: D = -16.162(6) cm(-1); E = -3.694(4) cm(-1); g(x) = 1.86(1); g(y) = 1.90(1); g(z) = 1.710(4). Analysis of the VTVH-MCD data gave spin Hamiltonian parameters in good agreement with those determined by HFEPR for both V(acac)(3) and VBr(3)(thf)(3) and in rough agreement with the estimate for VCl(3)(thf)(3) (D approximately 10 cm(-1), |E/D| approximately 0.18), together with the finding that the value of D is negative for both thf complexes. The electronic structures of these V(III) complexes are discussed in terms of their molecular structures and the electronic transitions observed by electronic absorption and MCD spectroscopies.     

Performance of an ultra-low elution-volume 96-well plate: drug discovery and development applications

Recently, sample preparation has been considered to be the major cause of bottlenecks during high-throughput analysis. With the assistance of robotic liquid handlers and the 96-well plate format, more samples can be prepared for subsequent liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Protein precipitation is still widely used despite potential loss of sensitivity or variable results due to ion suppression. The use of solid-phase extraction (SPE) clearly gives superior results but may not be as cost effective as protein precipitation due to the labor and material costs associated with the process. Here, a novel 96-well SPE plate is described that was designed to minimize the elution volume required for quantitative elution of analytes. The plate is packed with 2 mg of a high-capacity SPE sorbent that allows loading of up to 750 microL of plasma, while the novel design permits elution with as little as 25 microL. Therefore, the plate offers up to a 15-fold increase in sample concentration. The evaporation and reconstitution step that is typically required in SPE is avoided due to the concentrating ability of the plate. Examples of applications in drug discovery/development are shown and results are compared to protein precipitation. Excellent sensitivity and linearity are demonstrated.