Isotope-coded dimethyl tagging for differential quantification of posttranslational protein carbonylation by 4-hydroxy-2-nonenal, an end-product of lipid peroxidation

Peroxidation of cellular membrane lipids, rich in polyunsaturated fatty acids, generates electrophilic, α, β-unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE). HNE is a highly reactive and cytotoxic molecule that can react with the nucleophilic sites in proteins causing posttranslational modification. The identification of protein targets is an important first step; however, quantitative profiling of site-specific modifications is necessary to understand the biological impact of HNE-induced carbonylation. We report a method that uses light (H(12)CHO) and heavy (D(13)CDO) isotopic variant of formaldehyde to differentially label primary amines (N-termini and ε-amino group of lysines) in peptides through reductive methylation and, combined with selective enrichment of modified peptides, permits comparison of the extent of carbonylation in two samples after mixing for simultaneous liquid chromatography-mass spectrometry. Specifically, dimethyl-labeled peptide carbonyls were fractionated from unmodified peptides using solid-phase hydrazide chemistry to immobilize them to porous glass beads and, after removing the unmodified peptides by thoroughly washing the beads, subsequently recover them by acid-catalyzed hydrolysis. The method was developed using HNE-modified synthetic peptides and also showing enrichment from a complex matrix of digested human plasma proteins. Applicability was confirmed using apomyoglobin as an analyte, implicating thereby its potential value to proteome-wide identification and relative quantification of posttranslational protein carbonylation with residue-specific information. Because HNE attachment may not necessarily cause change in protein abundance, this modification-focused quantification should facilitate the characterization of accompanied changes in protein function and, also, provide important insights into molecular signaling mechanisms and a better understanding of cellular processes associated with oxidative stress.     

Fourier transform ion cyclotron resonance mass spectrometry of covalent adducts of proteins and 4-hydroxy-2-nonenal, a reactive end-product of lipid peroxidation

Covalent adduction of the model protein apomyoglobin by 4-hydroxy-2-nonenal, a reactive end-product of lipid peroxidation, was characterized by nanoelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The high mass resolving power and mass measurement accuracy of the instrument facilitated a detailed compositional analysis of the complex reaction product without the need for deconvolution and transformation to clearly show the pattern of adduction and component molecular weights. Our study has also demonstrated the value of electron capture dissociation over collision-induced dissociation for the tandem mass spectrometric determination of site modification for the 4-hydroxy-2-nonenal adduct of oxidized insulin B chain as an example. Figure FTICR allowed characterization of 4-hydroxy-2-nonenal (HNE)-modified apomyoglobin (an expanded spectrum of the +15 charge state is shown)     

Carboxymethylpsyllium (CMPsy)/poly(acrylamide)(poly(AAm)) hydrogels for oral delivery of anti-diabetic drug Gliclazide

In this work, controlled release of anti-diabetic drug Gliclazide (Glz) from the carboxymethylpsyllium (CMPsy)/poly(acrylamide) (poly (AAm)) semi-interpenetrating networks (Semi-IPN) has been studied under physiological conditions. The semi-IPN was characterized by TGA, XRD and FTIR analysis. The release of drug Glz was observed to increase with concentration of drug entrapped in the hydrogels but varied inversely with the degree of crosslinking of the semi-IPN. The release data were analyzed using various kinetic models. Finally, the in-vivo investigation of efficacy of Gliclazide–loaded semi-IPN was carried out on Albino wistar rats. The formulation demonstrated fair effectiveness in reducing the glucose level, even at an oral administration frequency of once every alternate day.     

Fourier decompositions and pulse sequence design algorithms for nuclear magnetic resonance in inhomogeneous fields

In this paper, we introduce algorithms based on Fourier synthesis for designing phase compensating rf pulse sequences for high-resolution nuclear magnetic resonance (NMR) spectroscopy in an inhomogeneous B0 field. We show that using radio frequency pulses and time varying linear gradients in three dimensions, it is possible to impart the transverse magnetization of spins phase, which is a desired function of the spatial (x,y,z) location. Such a sequence can be used to precompensate the phase that will be acquired by spins at different spatial locations due to inhomogeneous magnetic fields. With this precompensation, the chemical shift information of the spins can be reliably extracted and high resolution NMR spectrum can be obtained.     

Rapid characterization of covalent modifications to rat brain mitochondrial proteins after ex vivo exposure to 4-hydroxy-2-nonenal by liquid chromatography-tandem mass spectrometry using data-dependent and neutral loss-driven MS3 acquisition

The modification of mitochondrial proteins enriched from rat forebrain by the major lipid peroxidation product 4-hydroxy-2-nonenal (HNE) was investigated using high performance liquid chromatography (HPLC) and tandem mass spectrometry. Subcellular fractionation in conjunction with a 'shotgun-based' approach that involved both conventional data-dependent and neutral loss (NL)-driven MS(3) data acquisition on a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT) was utilized. Using a relatively rapid linear HPLC gradient (1 h) for complex mixture analysis, 24 sites of HNE modification on 15 unique proteins were identified which corresponded exclusively to Michael adduct formation on histidine residues. Since a number of HNE-modified peptides produced a predominant HNE NL fragment-ion signal upon collision-induced dissociation (CID), NL-driven MS(3) data-dependent acquisition was a valuable method to enhance fragmentation information for these particular modified peptides. Of the 24 HNE modification sites identified, approximately 25% were determined from the MS(3) spectra alone. We envision the reported methodology as an efficient screening approach for HNE modification site selectivity that could ultimately provide a foundation for the development of targeted schemes for the characterization of in vivo HNE-protein adducts.     

Optimal control based NCO and NCA experiments for spectral assignment in biological solid-state NMR spectroscopy

We present novel pulse sequences for magic-angle-spinning solid-state NMR structural studies of (13)C,(15)N-isotope labeled proteins. The pulse sequences have been designed numerically using optimal control procedures and demonstrate superior performance relative to previous methods with respect to sensitivity, robustness to instrumental errors, and band-selective excitation profiles for typical biological solid-state NMR applications. Our study addresses specifically (15)N to (13)C coherence transfers being important elements in spectral assignment protocols for solid-state NMR structural characterization of uniformly (13)C,(15)N-labeled proteins. The pulse sequences are analyzed in detail and their robustness towards spin system and external experimental parameters are illustrated numerically for typical (15)N-(13)C spin systems under high-field solid-state NMR conditions. Experimentally the methods are demonstrated by 1D (15)N-->(13)C coherence transfer experiments, as well as 2D and 3D (15)N,(13)C and (15)N,(13)C,(13)C chemical shift correlation experiments on uniformly (13)C,(15)N-labeled ubiquitin.     

Fabrication and testing of the recoil mass spectrometer at Bombay Pelletron

A recoil mass spectrometer (RMS) has been designed, fabricated and installed at the 15° S beam-line of the Pelletron at TIFR. The RMS consists of a quadrupole doublet just after the target chamber followed by an ‘electrostatic deflector’, a magnetic dipole and a second electrostatic deflector. The recoils produced in the ¹²C + ⁵⁸Ni reaction using 60 MeV ¹²C beam were focussed with the help of electric and magnetic fields and detected in a strip detector placed at the focal plane of the RMS. Further testing of the spectrometer to obtain mass resolution and efficiency are in progress.     

Locally Rotationally Symmetric Bianchi Type-Ⅰ Model with Time Varying Λ Term

We investigate the locally rotationally symmetric （LRS） Bianchi type-Ⅰ cosmological model for stiff matter and a vacuum solution with a cosmological term proportional to R^-m （R is the scale factor and m is a positive constant）. The cosmological term decreases with time. We obtain that for both the cases the present universe is accelerating with a large fraction of cosmological density in the form of a cosmological term.     

Facile synthesis of isatins by direct oxidation of indoles and 3-iodoindoles using NIS/IBX

A facile one-pot access to a broad range of isatins by direct oxidation of indoles using NIS/IBX reagent in DMSO at 25?°C in very good isolated yields is reported. It is shown by mechanistic investigations that a number of substituted indoles react rapidly with NIS in DMSO to produce intermediary 3-iodoindoles, which undergo oxidation subsequently to isatins with IBX.