The reactivity of human serum albumin toward trans‐4‐hydroxy‐2‐nonenal

Mass spectrometry was used to probe the preferred locations of trans‐4‐hydroxy‐2‐nonenal (HNE) addition to the cysteine, histidine, and lysine residues of human serum albumin (HSA). Considering only those modified peptides supported by high mass accuracy Orbitrap precursor ion measurements (high confidence hits), with HNE:HSA ratios of 1:1 and 10:1, 3 and 15 addition sites, respectively, were identified. Using less stringent criteria, a total of 34 modifications were identified at the higher concentration. To gain quantitative data, iTRAQ labeling studies were completed. Previous work had identified Cys³⁴, the only free cysteine, as the most reactive residue in HSA, and we have found that Lys¹⁹⁹, His^242/7, and His²⁸⁸ are the next most reactive residues. Although the kinetic data indicate that the lysines and histidines can react at relatively similar rates, the results show that lysine addition is much less favorable thermodynamically; under our reaction conditions, lysine addition generally does not go to completion. This suggests that under physiological conditions, HNE addition to lysine is only relevant in situations where unusually high HNE concentrations or access to irreversible secondary reactions are found. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification of carbonylation sites in apomyoglobin after exposure to 4‐hydroxy‐2‐nonenal by solid‐phase enrichment and liquid chromatography–electrospray ionization tandem mass spectrometry

Identification of protein carbonylation because of covalent attachment of a lipid peroxidation end‐product was performed by combining proteolytic digestion followed by solid‐phase hydrazide enrichment and liquid chromatography (LC)–electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using both collision‐induced dissociation (CID) and electron capture dissociation (ECD). To evaluate this approach, we selected apomyoglobin and 4‐hydroxy‐2‐nonenal (4‐HNE) as a model protein and a representative end‐product of lipid peroxidation, respectively. Although the characteristic elimination of 4‐HNE (156 Da) in CID was found to serve as a signature tag for the modified peptides, generation of nearly complete fragment ion series because of efficient peptide backbone cleavage (in most cases over 75%) and the capability to retain the labile 4‐HNE moiety of the tryptic peptides significantly aided the elucidation of primary structural information and assignment of exact carbonylation sites in the protein, when ECD was employed. We have concluded that solid‐phase enrichment with both CID‐ and ECD‐MS/MS are advantageous during an in‐depth interrogation and unequivocal localization of 4‐HNE‐induced carbonylation of apomyoglobin that occurs via Michael addition to its histidine residues. Copyright © 2010 John Wiley & Sons, Ltd.     

Mass spectrometry‐based proteomics of oxidative stress: Identification of 4‐hydroxy‐2‐nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins

Modification of proteins by 4‐hydroxy‐2‐nonenal (HNE), a reactive by‐product of ω6 polyunsaturated fatty acid oxidation, on specific amino acid residues is considered a biomarker for oxidative stress, as occurs in many metabolic, hereditary, and age‐related diseases. HNE modification of amino acids can occur either via Michael addition or by formation of Schiff‐base adducts. These modifications typically occur on cysteine (Cys), histidine (His), and/or lysine (Lys) residues, resulting in an increase of 156 Da (Michael addition) or 138 Da (Schiff‐base adducts), respectively, in the mass of the residue. Here, we employed biochemical and mass spectrometry (MS) approaches to determine the MS “signatures” of HNE‐modified amino acids, using lysozyme and BSA as model proteins. Using direct infusion of unmodified and HNE‐modified lysozyme into an electrospray quadrupole time‐of‐flight mass spectrometer, we were able to detect up to seven HNE modifications per molecule of lysozyme. Using nanoLC‐MS/MS, we found that, in addition to N‐terminal amino acids, Cys, His, and Lys residues, HNE modification of arginine (Arg), threonine (Thr), tryptophan (Trp), and histidine (His) residues can also occur. These sensitive and specific methods can be applied to the study of oxidative stress to evaluate HNE modification of proteins in complex mixtures from cells and tissues under diseased versus normal conditions.     

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.     

Mass spectrometric characterization of covalent modification of human serum albumin by 4-hydroxy-trans-2-nonenal

Several pieces of evidence indicate that albumin modified by HNE is a promising biomarker of systemic oxidative stress and that HNE-modified albumin may contribute to the immune reactions triggered by lipid peroxidation-derived antigens. In this study, we found by HPLC analysis that HNE is rapidly quenched by human serum albumin (HSA) because of the covalent adduction to the different accessible nucleophilic residues of the protein, as demonstrated by electrospray ionization mass spectrometry (ESI-MS) direct infusion experiments (one to nine HNE adducts, depending on the molar ratio used, from 1:0.25 to 1:5 HSA:HNE). An LC-ESI-MS/MS approach was then applied to enzymatically digested HNE-modified albumin, which permitted the identification of 11 different HNE adducts, 8 Michael adducts (MA) and 3 Schiff bases (SB), involving nine nucleophilic sites, namely: His67 (MA), His146 (MA), His242 (MA), His288 (MA), His510 (MA), Lys 195 (SB), Lys 199 (MA, SB), Lys525 (MA, SB) and Cys34 (MA). The most reactive HNE-adduction site was found to be Cys34 (MA) followed by Lys199, which primarily reacts through the formation of a Schiff base, and His146, giving the corresponding HNE Michael adduct. These albumin modifications are suitable tags of HNE-adducted albumin and could be useful biomarkers of oxidative and carbonylation damage in humans.     

Detection and quantification of 8‐hydroxy‐2′‐deoxyguanosine in Alzheimer's transgenic mouse urine using capillary electrophoresis

8‐Hydroxy‐2′‐deoxyguanosine (8‐OHdG) is one of the major forms of oxidative DNA damage, and is commonly analyzed as an excellent marker of DNA lesions. The purpose of this study was to develop a sensitive method to accurately and rapidly quantify the 8‐OHdG by using CE‐LIF detection. The method involved the use of specific antibody to detect the DNA lesion (8‐OHdG) and consecutive fluorescence labeling. Next, urinary 8‐OHdG fluorescently labeled along with other constituents were resolved by capillary electrophoretic system and the lesion of interest was detected using a fluorescence detector. The limit of detection was 0.18 fmol, which proved sufficient sensitivity for detection and quantification of 8‐OHdG in untreated urine samples. The relative standard deviation was found to be 11.32% for migration time and 5.52% for peak area. To demonstrate the utility of this method, the urinary concentration of 8‐OHdG in an Alzheimer's transgenic mouse model was determined. Collectively, our results indicate that this methodology offers great advantages, such as high separation efficiency, good selectivity, low limit of detection, simplicity and low cost of analysis.     

两类三组份反应产物：1,3-恶嗪及嘧啶-2-酮类化合物的质谱学行为研究

电喷雾质谱被应用于分辨2-氨基-1,3-恶嗪及六氢化-4-苯基-吡喃[2,3-d]嘧啶-2-酮的杂环结构。两类化合物均为三组份反应的产物,且其杂环的结构很难用NMR判断。实验首次系统研究了两类化合物的质谱学行为（包括氘代实验和高分辨质谱研究）,发现前者在CID实验中丢失CH2N2和HCNO,而后者为直接丢失尿素。这些特征丢失为该类衍生物的结构判断,尤其是高通量的合成产物分析提供了重要的依据。     

A Simple Synthesis of 2‐{(Arylmethylidene)hydrazinylidene]‐3‐hydroxy‐4H‐furo[3,2‐c]pyran‐4(3H)‐ones

A simple synthesis of 2‐hydrazinylidene‐3‐hydroxy‐4H‐furo[3,2‐c]pyran‐4‐ones is described. A mixture of (isocyanoimino)(triphenyl)phosphorane, an aromatic aldehyde, and dehydroacetic acid (=3‐acetyl‐2‐hydroxy‐6‐methyl‐4H‐pyran‐4‐one) undergo a 1 : 1 : 1 addition reaction under mild conditions to afford the title compounds in excellent yields.     

Polymorphism in 3‐acetyl‐4‐hydroxy‐2H‐chromen‐2‐one

A new polymorph (denoted polymorph II) of 3‐acetyl‐4‐hydroxy‐2H‐chromen‐2‐one, C₁₁H₈O₄, was obtained unexpectedly during an attempt to recrystallize the compound from salt–melted ice, and the structure is compared with that of the original polymorph (denoted polymorph I) [Lyssenko & Antipin (2001). Russ. Chem. Bull. 50 , 418–431]. Strong intramolecular O—H...O hydrogen bonds are observed equally in the two polymorphs [O...O = 2.4263 (13) Å in polymorph II and 2.442 (1) Å in polymorph I], with a slight delocalization of the hydroxy H atom towards the ketonic O atom in polymorph II [H...O = 1.32 (2) Å in polymorph II and 1.45 (3) Å in polymorph I]. In both crystal structures, the packing of the molecules is dominated and stabilized by weak intermolecular C—H...O hydrogen bonds. Additional π–π stacking interactions between the keto–enol hydrogen‐bonded rings stabilize polymorph I [the centres are separated by 3.28 (1) Å], while polymorph II is stabilized by interactions between α‐pyrone rings, which are parallel to one another and separated by 3.670 (5) Å.     

Distinguishing of 2‐MAPB and 6‐MAPB: Solution of the problem

Differentiation of new psychoactive substance (NPS), 6‐(2‐methylaminopropyl)benzofuran (6‐MAPB), and its positional isomer, 2‐(2‐methylaminopropyl)benzofuran (2‐MAPB), by means of gas chromatography/mass spectrometry (GC/MS) with quadrupole detection is ambiguous. Reliable distinguishing of the two isomers could be achieved by MS/MS spectra recorded after collision‐induced dissociation (CID) of precursor ions. Both electron ionization (EI) and electrospray ionization (ESI) methods could be used for these purposes.     

Incubation with Cu(II) and Zn(II) salts enhances MALDI‐TOF mass spectra of amyloid‐beta and α‐synuclein toward in vivo analysis

Insoluble senile plaque aggregates are indicative of Alzheimer's disease pathology. A similar phenomenon occurs in Parkinson's disease with the build‐up of Lewy bodies. The analysis of senile plaques, and other brain samples, from Alzheimer's disease and Parkinson's disease patients by matrix‐assisted laser desorption/ionization mass spectrometry has advantages but also presents obstacles because of the nature of the processes utilized in isolation procedures and storage. Salts, buffers, and detergents necessary in the isolation of biological species may cause adducts and ion suppression that convolute the spectra obtained. We previously determined that amyloid‐beta from isolated senile plaque deposits fragment similarly to the synthetic 40 and 42 amino acid peptide when analyzed by matrix‐assisted laser desorption/ionization mass spectrometry. In addition, α‐synuclein also fragments predictably by in‐source decay. This provides information that may be applied to the identification and localization of amyloid‐beta and α‐synuclein in senile plaques and intact tissue sections. Ion suppression must still be accounted for when analyzing biological samples, which makes identifying fragments at lower abundance difficult. The addition of certain transition‐metal salts (Cu(II), Zn(II)) to the sample prior to analysis serves to “clean” the spectra and allow the peptide fragments produced to be observed with a much higher signal to noise and occasionally, improved resolution. We present a systematic study of incubation with different metal salts and their impact on the quality of the spectra, as well as the role of the binding of the metals to the model biological compounds, obtained for synthetic amyloid‐beta, synthetic α‐synuclein, and isolated senile plaques. The optimized sample preparation methods presented will provide for simpler and more thorough identification of these biologically relevant species in human‐derived samples.     

Facile Synthesis of Substituted Ethyl 2‐(Chloromethyl)‐2‐hydroxy‐2H‐1‐benzopyran‐3‐carboxylates

Ethyl 2‐(chloromethyl)‐2‐hydroxy‐2H‐chromene‐3‐carboxylates 2a – 2j have been synthesized by reaction of substituted salicylaldehydes with ethyl 4‐chloro‐3‐oxobutanoate, in the presence of piperidine in CH₂Cl₂ at room temperature, in good yields.     

The chromatographic assay of 4‐hydroxynonenal as a biomarker of diseases by means of MEPS and HPLC technique

The aim of this study was to develop and validate a new analytical method for the determination of 4‐hydroxy‐2‐nonenal (4‐HNE) in biological samples while applying microextraction by packed sorbent as a sample preparation method and HPLC with UV–vis detection. Various microextraction by packed sorbent (MEPS) sorbents like C₂, C₈, C₁₈, M1 (80% C₈ and 20% SCX) and silica were used to separate 4‐HNE from biological samples. The highest affinity of 4‐HNE was observed for sorbents like C₁₈. The extraction efficiency was in the range from 47.4 to 89.2% dependent on the concentration of 4‐HNE. Lower efficiency of 4‐HNE extraction was obtained with use of MEPS packings such as C₈ and M1. The extraction efficiency was in the range from 35.2 to 66.3% for packing C8 and from 34.2 to 64.3% for packing M1, respectively. The limit of detection and lower limit of quantitation for UV–vis detection were respectively 4.5 and 9.0 nmol/mL. The proposed method can be used for the evaluation of extraction efficiency of 4‐HNE in biological sample because the values of lower limit of quantitation are lower than the determined amounts of the analyte in samples. The method yields excellent performance of quantification and identification in analysis of inflammation biomarkers. Copyright © 2014 John Wiley & Sons, Ltd.     

A mass spectrometric analysis of 4-hydroxy-2-(E)-nonenal modification of cytochrome c

Cytochrome c is a key mitochondrial respiratory protein that is particularly susceptible to modification during oxidative stress. The nature of this susceptibility is linked to the mitochondrial membrane being rich in esterified linoleic acid, which predisposes this organelle to the formation of lipid peroxidation products such as 4-hydroxy-2-(E)-nonenal (4-HNE). To better understand the nature of cytochrome c modification by 4-HNE, we initiated an in vitro study utilizing a combination of MALDI-TOF mass spectrometry, LC-ESI-MS/MS and isotope labeling to monitor 4-HNE modification of cytochrome c under various conditions. The overwhelming reaction observed is Michael addition by Lys side-chains in addition to the modification of His 33. While the Lys-4-HNE adducts were generally observed to be reversible, the 4-HNE-His 33 was observed to be stable with half of the formed adduct surviving the denaturation and proteolysis protocols used to generate proteolytic peptides for LC-ESI-MS/MS.     

Simultaneous analysis of 2‐methylimidazole, 4‐methylimidazole,and 5‐hydroxymethylfurfural potentially formed in fermented soy sauce by “quick,easy, cheap,effective, rugged,and safe” purification and UHPLC with tandem mass spectrometry

2‐Methylimidazole, 4‐methylimidazole and 5‐hydroxymethylfurfural are harmful by‐products potentially formed via Maillard reaction in fermented soy sauce. The present study proposed a new method based on “quick, easy, cheap, effective, rugged, and safe” purification and ultra high performance liquid chromatography with tandem mass spectrometry for the simultaneous analysis of 2‐methylimidazole, 4‐methylimidazole and 5‐hydroxymethylfurfural in fermented soy sauce. The sample was dissolved in water after addition of internal standard 4‐methylimidazole‐d₆ and extracted with acetonitrile. After dehydration, it was centrifuged and the supernatant was subsequently purified using two sorbents namely primary‐secondary amine and multi‐walled carbon nanotube. Three target analytes were separated by gradient elution and determined under multiple reactions monitoring mode. The limit of detection, matrix effect, recovery and precision of the developed method were investigated. Results found that three target analytes displayed excellent linearity in concentration range of 1–250 μg/L. Limit of detection was in the range of 0.3–1 μg/kg for three target analytes. The mean recoveries for fermented soy sauce samples at three spiked concentrations were in the range of 91.2–112.5%, and the intra‐ and interday precision were in the ranges of 3.6–9.2 and 7.1–10.8%, respectively. This validated method was successfully applied to determine 2‐methylimidazole, 4‐methylimidazole and 5‐hydroxymethylfurfural concentrations in fermented soy sauce.     

The solid‐state emmissive chalcone (2E)‐1‐(5‐chlorothiophen‐2‐yl)‐3‐[4‐(dimethylamino)phenyl]prop‐2‐en‐1‐one

Orange rectangular blocks suitable for X‐ray diffraction analysis were obtained for the previously reported [Ahmad & Bano (2011). Int. J. ChemTech Res. 3 , 1470–1478] title chalcone, C₁₅H₁₄ClNOS. This solid‐emissive chalcone exhibits a planar structure and the bond parameters are compared with related compounds already described in the literature. The determination of the structure of this chalcone is quite relevant because it will play an important role in theoretical calculations to investigate potential two‐photon absorption processes and could also be useful for studying the interaction of such compounds with a biological target.     

Regioselective Synthesis of 4‐(Arylsulfanyl)‐2‐hydroxyhomophthalates by [4+2] Cycloaddition of 3‐(Arylsulfanyl)‐1‐(trimethylsilyloxy)buta‐1,3‐dienes with Dimethyl Penta‐2,3‐dienedioate

The [4+2] cycloaddition of 3‐(arylsulfanyl)‐1‐(trimethylsilyloxy)buta‐1,3‐dienes with dimethyl penta‐2,3‐dienedioate provides a convenient and regioselective approach to a variety of 4‐(arylsulfanyl)‐2‐hydroxyhomophthalates.     

Heterocyclic ring cleavage upon collision‐induced dissociation of deprotonated 3‐hydroxy‐1,2,5‐oxadiazoles (3‐hydroxyfurazans)

A series of 4‐substituted 3‐hydroxyfurazans were subjected to electrospray ionization tandem mass spectrometry. At low collision energy, oxyisocyanate ([O=C=N–O]^?, m/z 58) was formed as the predominant product ion from each deprotonated 3‐hydroxyfurazan, indicating cleavage of the heterocyclic ring. The facile energetics of this characteristic fragmentation process was confirmed by density functional computations.     

A metabolomic study of the urine of rats with Alzheimer's disease and the efficacy of Ding‐Zhi‐Xiao‐Wan on the afflicted rats

As a well‐known traditional Chinese medicine formula, Ding‐Zhi‐Xiao‐Wan has long been used for the routine treatment of Alzheimer's disease. However, the mechanism of Ding‐Zhi‐Xiao‐Wan in treating Alzheimer's disease is unclear. Therefore, a nontargeted metabolomics method based on ultrahigh performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry has been established to explore the metabolic variations in the urine of Alzheimer's disease rats and investigate the therapeutic mechanism of Ding‐Zhi‐Xiao‐Wan on Alzheimer's disease. To develop a better rat model of Alzheimer's disease, amyloid β25‐35 was injected into the bilateral hippocampus of Sprague–Dawley rats. Multivariate analysis approaches were applied to differentiate the urine components between the four groups. Thereafter, a targeted metabolomics method was used to verify the identified endogenous metabolites and determine the mechanism of action of Ding‐Zhi‐Xiao‐Wan. Altogether, 26 potential biomarkers were found, of which 15 biomarkers (10 of which are potential biomarkers found in nontargeted metabolomics) were identified. The results show that Ding‐Zhi‐Xiao‐Wan mainly affects the pathways of taurine and hypotaurine metabolism, tryptophan metabolism, and phenylalanine metabolism. Ding‐Zhi‐Xiao‐Wan might play a role in the treatment of Alzheimer's disease by mediating antioxidative stress, regulation of energy metabolism, improvement of intestinal microbes, and protection of nerve cells.     

3‐Cyano‐6‐hydroxy‐4‐methyl‐2‐pyridone: two new pseudopolymorphs and two cocrystals with products of an in situ nucleophilic aromatic substitution

Four crystal structures of 3‐cyano‐6‐hydroxy‐4‐methyl‐2‐pyridone (CMP), viz. the dimethyl sulfoxide monosolvate, C₇H₆N₂O₂·C₂H₆OS, (1), the N,N‐dimethylacetamide monosolvate, C₇H₆N₂O₂·C₄H₉NO, (2), a cocrystal with 2‐amino‐4‐dimethylamino‐6‐methylpyrimidine (as the salt 2‐amino‐4‐dimethylamino‐6‐methylpyrimidin‐1‐ium 5‐cyano‐4‐methyl‐6‐oxo‐1,6‐dihydropyridin‐2‐olate), C₇H₁₃N₄⁺·C₇H₅N₂O₂⁻, (3), and a cocrystal with N,N‐dimethylacetamide and 4,6‐diamino‐2‐dimethylamino‐1,3,5‐triazine [as the solvated salt 2,6‐diamino‐4‐dimethylamino‐1,3,5‐triazin‐1‐ium 5‐cyano‐4‐methyl‐6‐oxo‐1,6‐dihydropyridin‐2‐olate–N,N‐dimethylacetamide (1/1)], C₅H₁₁N₆⁺·C₇H₅N₂O₂⁻·C₄H₉NO, (4), are reported. Solvates (1) and (2) both contain the hydroxy group in a para position with respect to the cyano group of CMP, acting as a hydrogen‐bond donor and leading to rather similar packing motifs. In cocrystals (3) and (4), hydrolysis of the solvent molecules occurs and an in situ nucleophilic aromatic substitution of a Cl atom with a dimethylamino group has taken place. Within all four structures, an R₂²(8) N—H...O hydrogen‐bonding pattern is observed, connecting the CMP molecules, but the pattern differs depending on which O atom participates in the motif, either the ortho or para O atom with respect to the cyano group. Solvents and coformers are attached to these arrangements via single‐point O—H...O interactions in (1) and (2) or by additional R₄⁴(16) hydrogen‐bonding patterns in (3) and (4). Since the in situ nucleophilic aromatic substitution of the coformers occurs, the possible Watson–Crick C–G base‐pair‐like arrangement is inhibited, yet the cyano group of the CMP molecules participates in hydrogen bonds with their coformers, influencing the crystal packing to form chains.