Robust analysis of angiotensin peptides in human plasma: Column switching-parallel LC/ESI-SRM/MS without adsorption or enzymatic decomposition.

Angiotensin (Ang) peptides are the main effectors of the renin-angiotensin system (RAS) regulating diverse physiological conditions and are involved in renal and vascular diseases. Currently, quantitative analyses of Ang peptides in human plasma mainly rely on radioimmunoassay-based methods whose reported levels are quite divergent. Analyses are further complicated by the potential of Ang peptides to bind to solid surfaces, to be enzymatically decomposed during sample preparation, and to undergo post-translational modifications. A column switching-parallel LC/ESI-SRM/MS method has been developed for seven Ang peptides (Ang I, Ang II, Ang III, Ang IV, Ang 1-9, Ang 1-7, and Ang A) in human plasma. Aqueous acetonitrile (5%) containing 50 mM arginine (Arg) as a dissolving solution and a combination of protease inhibitors with formic acid were used to prevent adsorption and enzymatic degradation, respectively. Plasma samples were simply deproteinized with acetonitrile followed by clean-up with an on-line trap column via column-switching. Stable isotope dilution with [13C5,15N1-Val]-Ang peptides as internal standards was employed for quantitative analysis. The current methodology has been successfully applied to determine the plasma levels of Ang peptides in healthy participants, suggesting future applicability to studies of various diseases related to RAS.

Screening of Chemical Modifications in Human Skin Keratins by Mass Spectrometry-Based Proteomic Analysis via Noninvasive Sampling and On-Tape Digestion.

Proteins are continuously exposed to diverse chemical stresses, and the resulting chemical modifications can provide significant information on biological events. Keratins are the main constituent of human skin and are the major target proteins of various chemical modifications. We have previously developed a mass spectrometry-based noninvasive proteomic methodology to screen oxidative modifications in human skin keratins. We have improved this methodology in terms of sample preparation time and amino acid sequence coverage using an on-tape digestion method. After sampling by tape stripping, skin proteins on the tape were subjected to reduction/alkylation, followed by trypsin digestion without a presolubilization step using detergents. To screen chemical modifications in keratins, target modifications and tryptic target peptides carrying the modification sites were determined from in vitro experiments with major reactive chemical species (4-hydroxy-2(E)-nonenal (HNE), 4-oxo-2(E)-nonenal, glucose, methylglyoxal, peroxynitrite, and hydrogen peroxide). The developed method was used to screen target modifications in controls and patients with a swollen red rash. Basal levels of lipid-derived modification, oxidation, nitration, and glycation in keratins were detected in controls. Principal component analysis based on the relative chemical modification resulted in a clear classification of both groups within a 95% confidence interval. Lipid-derived HNE modification increased most significantly in the patient group. This methodology can be easily applied to patients with other diseases, and the target modifications can be used as biomarkers of certain physiological conditions.

Angiotensin II-Induced Oxidative Stress in Human Endothelial Cells: Modification of Cellular Molecules through Lipid Peroxidation.

Angiotensin (Ang) II is a major bioactive peptide of the renin/angiotensin system and is involved in various cardiovascular functions and diseases. Ang II type 1 (AT1) receptor mediates most of the physiological effects of Ang II. Previous studies have revealed that the lipid peroxidation products 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE) readily modify the N-terminus and Asp1, Arg2, and His6 residues of Ang II, and these modifications alter the biological activities of Ang II. Ang II is known to stimulate the formation of reactive oxygen species (ROS) that mediate cardiovascular remodeling. Another major consequence of ROS-derived damage is lipid peroxidation, which generates genotoxic aldehydes such as ONE and HNE. This study demonstrated that Ang II induced lipid peroxidation-derived modifications of cellular molecules in EA.hy926 cells, a human vascular endothelial cell line. Ang P (ONE- and ROS-derived N-terminal pyruvamide Ang II) and [His6(HNE)]-Ang II were detected in the medium of EA.hy926 cells incubated with Ang II, and their concentrations increased dose-dependently upon the addition of ascorbic acid (AscA) and CuSO4. Cells were then subjected to metabolic labeling using SILFAC (stable isotope labeling by fatty acids in cell culture) with [13C18]-linoleic acid. Analysis of cellular phospholipids indicated over 90% labeling. [13C9]-Thiadiazabicyclo-ONE-glutathione adduct as well as Ang P and [His6([13C9]-HNE)]-Ang II was detected in the labeled cells upon treatment with Ang II and their concentrations increased in an Ang II dose-dependent manner. Incubation of the labeled cells with losartan, an AT1 receptor blocker, inhibited the formation of modified Ang IIs in a dose-dependent manner. These results indicate that Ang II induces lipid peroxidation and modification of various cellular molecules and these reactions are mediated by the activation of AT1 receptor. Therefore, lipid peroxidation could be one mechanism by which Ang II contributes to cardiovascular dysfunction.

Imidazole dipeptides can quench toxic 4-oxo-2(E)-nonenal: Molecular mechanism and mass spectrometric characterization of the reaction products.

Imidazole dipeptides, such as carnosine (ß-alanyl-l-histidine) and anserine (ß-alanyl-Nπ -methyl-l-histidine), are highly localized in excitable tissues, including skeletal muscle and nervous tissue, and play important roles such as scavenging reactive oxygen species and quenching reactive aldehydes. We have demonstrated several reactions between imidazole dipeptides (namely, carnosine, and anserine) and a lipid peroxide-derived reactive aldehyde 4-oxo-2(E)-nonenal. Seven carnosine adducts and two anserine adducts were characterized using liquid chromatography/electrospray ionization-multiple-stage mass spectrometry. Adduct formation occurred between imidazole dipeptides and 4-oxo-2(E)-nonenal mainly through Michael addition, Schiff base formation, and/or Paal-Knorr reaction. The reactions were much more complicated than the reaction with a similar lipid peroxide-derived reactive aldehyde, 4-hydroxy-2(E)-nonenal.

Biomimetic trapping cocktail to screen reactive metabolites: use of an amino acid and DNA motif mixture as light/heavy isotope pairs differing in mass shift.

Candidate drugs that can be metabolically transformed into reactive electrophilic products, such as epoxides, quinones, and nitroso compounds, are of special concern because subsequent covalent binding to bio-macromolecules can cause adverse drug reactions, such as allergic reactions, hepatotoxicity, and genotoxicity. Several strategies have been reported for screening reactive metabolites, such as a covalent binding assay with radioisotope-labeled drugs and a trapping method followed by LC-MS/MS analyses. Of these, a trapping method using glutathione is the most common, especially at the early stage of drug development. However, the cysteine of glutathione is not the only nucleophilic site in vivo; lysine, histidine, arginine, and DNA bases are also nucleophilic. Indeed, the glutathione trapping method tends to overlook several types of reactive metabolites, such as aldehydes, acylglucuronides, and nitroso compounds. Here, we introduce an alternate way for screening reactive metabolites as follows: A mixture of the light and heavy isotopes of simplified amino acid motifs and a DNA motif is used as a biomimetic trapping cocktail. This mixture consists of [2H0]/[2H3]-1-methylguanidine (arginine motif, Δ 3 Da), [2H0]/[2H4]-2-mercaptoethanol (cysteine motif, Δ 4 Da), [2H0]/[2H5]-4-methylimidazole (histidine motif, Δ 5 Da), [2H0]/[2H9]-n-butylamine (lysine motif, Δ 9 Da), and [13C0,15N0]/[13C1,15N2]-2'-deoxyguanosine (DNA motif, Δ 3 Da). Mass tag triggered data-dependent acquisition is used to find the characteristic doublet peaks, followed by specific identification of the light isotope peak using MS/MS. Forty-two model drugs were examined using an in vitro microsome experiment to validate the strategy. Graphical abstract Biomimetic trapping cocktail to screen reactive metabolites.

Quantitative LC/ESI-SRM/MS of antibody biopharmaceuticals: use of a homologous antibody as an internal standard and three-step method development.

Monoclonal antibody-based therapeutic agents (antibody drugs) have attracted considerable attention as a new type of drug. Concomitantly, the use of quantitative approaches for characterizing antibody drugs, such as liquid chromatography (LC)-mass spectrometry (MS), has increased. Generally, selective quantification of antibody drugs is done using unique peptides from variable regions (V H and V L) as surrogate peptides. Further, numerous internal standards (ISs) such as stable isotope-labeled (SIL)-intact proteins and SIL-surrogate peptides are used. However, developing LC-MS methodology for characterizing antibody drugs is time-consuming and costly. Therefore, LC-MS is difficult to apply for this purpose, particularly during the drug discovery stage when numerous candidates must be evaluated. Here, we demonstrate an efficient approach to developing a quantitative LC/electrospray ionization (ESI)-selected reaction monitoring (SRM)/MS method for characterizing antibody drugs. The approach consists of the following features: (i) standard peptides or SIL-IS are not required; (ii) a peptide from the homologous monoclonal antibody serves as an IS; (iii) method development is monitored using a spiked plasma sample and one quantitative MS analysis; and (iv) three predicted SRM assays are performed to optimize quantitative SRM conditions such as transition, collision energy, and declustering potential values. Using this strategy, we developed quantitative SRM methods for infliximab, alemtuzumab, and bevacizumab with sufficient precision (<20%)/accuracy (<±20%) for use in the drug discovery stage. We have also demonstrated that choosing a higher homologous peptide pair (from analyte mAb/IS mAb) is necessary to obtain the sufficient precision and accuracy. Graphical abstract ᅟ.

An LC/ESI-SRM/MS method to screen chemically modified hemoglobin: simultaneous analysis for oxidized, nitrated, lipidated, and glycated sites.

Proteins are continuously exposed to various reactive chemical species (reactive oxygen/nitrogen species, endogenous/exogenous aldehydes/epoxides, etc.) due to physiological and chemical stresses, resulting in various chemical modifications such as oxidation, nitration, glycation/glycoxidation, lipidation/lipoxidation, and adduct formation with drugs/chemicals. Abundant proteins with a long half-life, such as hemoglobin (Hb, t 1/2 63 days, ∼150 mg/mL), are believed to be major targets of reactive chemical species that reflect biological events. Chemical modifications on Hb have been investigated mainly by mechanistic in vitro experiments or in vivo/clinical experiments focused on single target modifications. Here, we describe an optimized LC/ESI-SRM/MS method to screen oxidized, nitrated, lipidated, and glycated sites on Hb. In vivo preliminary results suggest that this method can detect simultaneously the presence of oxidation (+16 Da) of α-Met(32), α-Met(76), ß-Met(55), and ß-Trp(15) and adducts of malondialdehyde (+54 Da) and glycation (+162 Da) of ß-Val(1) in a blood sample from a healthy volunteer. Graphical Abstract Screening chemical modifications on hemoglobin.

Lgr4 controls specialization of female gonads in mice.

Leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is a type of membrane receptor with a seven-transmembrane structure. LGR4 is homologous to gonadotropin receptors, such as follicle-stimulating hormone receptor (Fshr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr). Recently, it has been reported that Lgr4 is a membrane receptor for R-spondin ligands, which mediate Wnt/beta-catenin signaling. Defects of R-spondin homolog (Rspo1) and wingless-type MMTV integration site family, member 4 (Wnt4) cause masculinization of female gonads. We observed that Lgr4(-/-) female mice show abnormal development of the Wolffian ducts and somatic cells similar to that in the male gonads. Lgr4(-/-) female mice exhibited masculinization similar to that observed in Rspo1-deficient mice. In Lgr4(-/-) ovarian somatic cells, the expression levels of lymphoid enhancer-binding factor 1 (Lefl) and Axin2 (Axin2), which are target genes of Wnt/beta-catenin signaling, were lower than they were in wild-type mice. This study suggests that Lgr4 is critical for ovarian somatic cell specialization via the cooperative signaling of Rspo1 and Wnt/beta-catenin.

Hydroxyl Radical-Mediated Novel Modification of Peptides: N-Terminal Cyclization through the Formation of α-Ketoamide.

The hydroxyl radical-mediated oxidation of peptides and proteins constitutes a large group of post-translational modifications that can result in structural and functional changes. These oxidations can lead to hydroxylation, sulfoxidation, or carbonylation of certain amino acid residues and cleavage of peptide bonds. In addition, hydroxyl radicals can convert the N-terminus of peptides to an α-ketoamide via abstraction of the N-terminal α-hydrogen and hydrolysis of the ketimine intermediate. In the present study, we identified N-terminal cyclization as a novel modification mediated by a hydroxyl radical. The reaction of angiotensin (Ang) II (DRVYIHPF) and the hydroxyl radical generated by the Cu(II)/ascorbic acid (AA) system or UV/hydrogen peroxide system produced N-terminal cyclized-Ang II (Ang C) and pyruvamide-Ang II (Ang P, CH3COCONH-RVYIHPF). The structure of Ang C was confirmed by mass spectrometry and comparison to an authentic standard. The subsequent incubation of isolated Ang P in the presence of Cu(II)/AA revealed that Ang P was the direct precursor of Ang C. The proposed mechanism involves the formation of a nitrogen-centered (aminyl) radical, which cyclizes to form a five-membered ring containing the alkoxy radical. The subsequent ß-scission reaction of the alkoxyl radical results in the cleavage of the terminal CH3CO group. The initial aminyl radical can be stabilized by chelation to the Cu(II) ions. The affinity of Ang C toward the Ang II type 1 receptor was significantly lower than that of Ang II or Ang P. Ang C was not further metabolized by aminopeptidase A, which converts Ang II to Ang III. Hydroxyl radical-mediated N-terminal cyclization was also observed in other Ang peptides containing N-terminal alanine, arginine, valine, and amyloid ß 1-11 (DAEFRHDSGYE).

Predicted multiple selected reaction monitoring to screen activated drug-mediated modifications on human serum albumin.

Metabolic activation of drugs frequently generates electrophilic products that may undergo covalent binding to biological macromolecules, such as proteins and DNA. The resulting covalent adducts are of considerable concern in drug discovery and development. Several strategies for assessing the potential risks of candidate drugs have been reported. Of these, glutathione trapping is the most commonly used method together with mass spectrometry. Furthermore, drug-mediated protein modifications have been studied using serum albumin and CYP enzymes to clarify target amino acids and mechanism-based inhibition, respectively. In this article, we introduce a practical way to screen drug-mediated protein modifications. The method, referred to as "predicted multiple selected reaction monitoring," is based on the selected reaction monitoring (SRM) strategy, but targets all possible chemically modified tryptic peptides. The creation of SRM lists may require patience; however, this strategy could facilitate more sensitive screening compared with the common strategy of data-dependent product ion scanning. Ketoprofen-N-hydroxysuccinimidyl ester (equivalent to glucuronide) and N-acetyl-p-benzoquinone imine (NAPQI) were allowed to react with human serum albumin as a model experiment. Using this strategy, 11 ketoprofen-adduction sites (at Lys(137, 195, 199, 212, 351, 402, 432, 436, 525, 536, and 541)) and 1 NAPQI-adduction site (at Cys(34)) were easily identified.

N-terminal α-ketoamide peptides: formation and transamination.

We have previously reported that N-terminal α-ketoamide peptides can be formed through 4-oxo-2(E)-nonenal (ONE)-derived oxidative decarboxylation of aspartic acid (Asp), which converts angiotensin (Ang) II (DRVYIHPF) to pyruvamide-Ang II (Ang P, CH3COCONH-RVYIHPF). The pyruvamide group significantly inhibits Ang P binding to the Ang II type 1 receptor, which mediates the major biological effects of Ang II. In the present study, we found that ONE can also introduce an α-ketoamide moiety at the N-terminus of peptides containing N-terminal residues other than Asp. Subsequent investigation of alternative biosynthetic pathways for N-terminal α-ketoamide peptides revealed that hydroxyl radical-mediated formation is a much more efficient route. The proposed mechanism involves initial abstraction of the N-terminal α-hydrogen and hydrolysis of the ketimine intermediate. The resulting N-terminal α-ketoamide is then converted to the D- and L-amino acids by nonenzymatic transamination in the presence of pyridoxamine (PM). The formation of the epimeric N-terminus depended on the incubation time and the concentration of PM, and increased further upon the addition of Cu(II) ions. A conversion of approximately 60% after three days of incubation was observed for Ang P. We propose that the reaction intermediate contains a prochiral α-carbon and is stabilized by the chelate effect of Cu(II) ions. The ONE- and hydroxyl radical-derived formation of N-terminal α-ketoamide and its transamination in the presence of PM were also observed in amyloid ß 1-11 (DAEFRHDSGYE), where the N-terminal Asp was converted to epimeric alanine. This suggests that these N-terminal modifications could occur in vivo and modulate the biological functions of peptides and proteins.

SKGQA, a Peptide Derived from the ANA/BTG3 Protein, Cleaves Amyloid-ß with Proteolytic Activity.

Despite the extensive research conducted on Alzheimer's disease (AD) over the years, no effective drug for AD treatment has been found. Therefore, the development of new drugs for the treatment of AD is of the utmost importance. We recently reported the proteolytic activities of JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMA), synthetic peptides of nine amino acids each, derived from the Box A region of Tob1 and ANA/BTG3 proteins, respectively. Furthermore, two components of ANA-TA9, ANA-YA4 (YRMI) at the C-terminus end and ANA-SA5 (SKGQA) at the N-terminus end of ANA-TA9, exhibited proteolytic activity against amyloid-ß (Aß) fragment peptides. In this study, we identified the active center of ANA-SA5 using AEBSF, a serine protease inhibitor, and a peptide in which the Ser residue of ANA-SA5 was replaced with Leu. In addition, we demonstrate the proteolytic activity of ANA-SA5 against the soluble form Aß42 (a-Aß42) and solid insoluble form s-Aß42. Furthermore, ANA-SA5 was not cytotoxic to A549 cells. These results indicate that ANA-SA5 is a promising Catalytide and a potential candidate for the development of new peptide drugs targeting Aß42 for AD treatment.

MALDI-TOF/MS-based label-free binding assay for angiotensin II type 1 receptor: application for novel angiotensin peptides.

Angiotensin (Ang) II is critically involved in multiple cardiovascular diseases mainly through the Ang II type 1 (AT1) receptor. Thus, AT1 receptor antagonists have been widely used for the treatment of hypertension and other cardiovascular conditions. However, most binding assays for AT1 receptor are based on radioactivity or fluorescent labeling, which present disadvantages such as radioactive waste production and/or changes to the binding characteristics of the labeled receptor or ligand. This article describes the first matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MS)-based label-free binding assay for AT1 receptor that can be performed in a high-throughput format. The affinity of competitors can be directly compared to that of Ang II by quantifying Ang II dissociated from the receptor-ligand complex using 50% methanol in water. The sensitivity and selectivity of MS analysis was improved by employing a stable-isotope ((13)C, (15)N)-labeled internal standard. This assay provided binding results equivalent to those obtained using other methods and revealed a significant decrease in the binding affinity of Ang II when the N-terminus underwent oxidative modification. The current setup of the assay can be easily adapted for other Ang receptors and would facilitate drug discovery studies targeting Ang receptors.

Aldehyde stress-mediated novel modification of proteins: epimerization of the N-terminal amino acid.

Various kinds of aldehyde-mediated chemical modifications of proteins have been identified as being exclusively covalent. We report a unique noncovalent modification: the aldehyde-mediated epimerization of the N-terminal amino acid. Epimerization of amino acids is thought to cause conformational changes that alter their biological activity. However, few mechanistic studies have been performed, because epimerization of an amino acid is a miniscule change in a whole protein. Furthermore, it does not produce a mass shift, making mass spectrometric analysis difficult. Here, we have demonstrated epimerization mediated by endogenous aldehydes. A model peptide, with an N-terminal l- or d-FMRFamide, was incubated with an endogenous or synthetic aldehyde [acetaldehyde, methylglyoxal, pyridoxal 5'-phosphate (PLP), 4-oxo-2(E)-nonenal, 4-hydroxy-2(E)-nonenal, d-glucose (Glc), 4- or 2-pyridinecarboxaldehyde] under physiological conditions. Each reaction mixture was analyzed by liquid chromatography with ultraviolet detection and/or electrospray ionization mass spectrometry. Considerable epimerization occurred after incubation with some endogenous aldehydes (PLP, 40.6% after 1 day; Glc with copper ions, 6.5% after 7 days). Moreover, the epimerization also occurred in whole proteins (human serum albumin and PLP, 26.3% after 1 day). Tandem mass spectrometric studies, including deuterium labeling and sodium borohydride reduction, suggested that the epimerization results from initial Schiff base formation followed by tautomerization to ketimine that causes the chirality to be lost. This suggests that the epimerization of the N-terminal amino acid can also occur in vivo as a post-translational modification under a high level of aldehyde stress.

Can Edman degradation be used for quantification? Isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry and the long-term stability of 20 phenylthiohydantoin-amino acids.

Edman degradation is a well-known method for obtaining amino acid (AA) sequences from a peptide by means of sequential reactions that release the N-terminal AAs from the peptide as a phenylthiohydantoin (PTH) derivative. Because of unexpected loss during the reaction and handling, there are few reports of use of this reaction for quantification. This manuscript describes the development of isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry for 20 PTH-AA derivatives, and long-term stability testing of PTH-AAs to ensure quantitative quality in the reaction. The 20 corresponding [(13)C6]-PTH-AAs were prepared by use of a one-pot reaction involving a mixture of [(13)C6]-Edman reagent and 20 AAs. Good linearity was observed for standard curves for the PTH-AAs, using the corresponding [(13)C6]-PTH-AAs as internal standards (1-100 pmol per injection, r(2) = 0.989-1.000). Serum albumin (human), pepsin (porcine stomach mucosa), α-casein (bovine milk), ribonuclease A (bovine), lysozyme (chicken egg white), and insulin (bovine) subjected to Edman degradation were examined as model proteins and peptides for N-terminal AA analysis. The results of the impurity test were satisfactory. Yield from the entire reaction with human serum albumin was estimated to be at least 75%, indicating great potential for absolute quantification of proteins without protein standards.

Complete amino acid sequencing and immunoaffinity clean-up can facilitate screening of various chemical modifications on human serum albumin.

This manuscript describes a simple and practical strategy for screening of various chemical modifications of human serum albumin (HSA). Serum albumin is the most abundant blood plasma protein in humans (HSA, 66.5 kDa, t1/2 = 19 d), constituting about 60 % of total proteins. Therefore, it is believed to be the main target of chemical stresses during physiological events such as increased oxidative stress from the degenerative diseases of aging, and higher glucose stress in diabetes mellitus. Consequently, chemical modifications can provide significant information about these biological events. In this study, a complete and robust sequencing method was attained by the peptide mass fingerprinting (PMF) technique using two different complementary proteases (trypsin and Glu-C) and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) in both positive and negative ionization modes. Using this strategy, several modified peptides, 12 oxidations, 25 glycations, 6 lipoxidations, and 5 nitrations have been identified on HSA treated with chemical reactions in vitro. Combined with immunoaffinity clean-up, this method was able to detect in vivo chemical modifications of HSA and found oxidized Trp(214) and glycated Lys(525) in healthy human plasma.

Lipid hydroperoxide-derived insulin resistance and its inhibition by pyridoxamine in skeletal muscle cells.

Oxidative stress is strongly associated with the onset and/or progression of diabetes. Under conditions of oxidative stress, lipid hydroperoxides are decomposed to reactive aldehydes that have been reported to induce insulin resistance by modifying proteins involved in insulin signaling. Pyridoxamine (PM) can inhibit the formation of advanced glycation/lipoxidation end products by scavenging reactive carbonyl species. Thus, PM has emerged as a promising drug candidate for various chronic conditions, including diabetic complications. In this study, L6 skeletal muscle cells were treated with 4-oxo-2(E)-nonenal (ONE), one of the most abundant and reactive lipid-derived aldehydes. Cellular insulin resistance was assessed by measuring insulin-stimulated glucose uptake using 2-deoxyglucose. ONE induced a time- and dose-dependent decrease in glucose uptake. Liquid chromatography/electrospray ionization-mass spectrometry analysis of the reaction between ONE and insulin receptor substrate 1 (IRS1) lysate identified multiple modifications that could disturb the interaction between IRS1 and activated IR, leading to insulin resistance. Pretreatment of the cells with PM restored the ONE-induced decrease in glucose uptake. Concomitantly, the formation of PM-ONE adducts in cell culture medium was increased in a PM-dose dependent manner. PM can therefore prevent lipid hydroperoxide-derived insulin resistance by quenching ONE. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-022-00155-z.

Lipid peroxidation-derived modification and its effect on the activity of glutathione peroxidase 1.

Oxidative stress and the resulting lipid peroxidation are associated with various pathological states, including neurodegenerative diseases and cancer. The end products of lipid peroxidation, such as 4-oxo-2(E)-nonenal (ONE), 4-hydroxy-2(E)-nonenal (HNE), and methylglyoxal (MG), exert several biological effects through modification of various cellular components, including DNA and proteins. Glutathione peroxidase 1 (GPx1) is an intracellular antioxidant enzyme that uses glutathione (GSH) to reduce a variety of peroxides, thereby modulating cellular oxidative stress and redox-mediated responses. GPx1 contains nucleophilic amino acids at its active (one Sec) and GSH-binding (four Arg and one Lys) sites. We found that lipid peroxidation-derived reactive aldehydes (ONE, HNE, and MG) modified the GSH-binding site, resulting in the inhibition of GPx1 activity. Mass spectrometry-based proteomic analysis identified the sites modified by each aldehyde (ONE, 14 sites; HNE, 7 sites; MG, 9 sites). The GSH-binding sites modified were as follows: ONE, Arg57, 103, 184, and 185; HNE, Lys91; MG, Arg103. Upon incubation of GPx1 with each aldehyde, ONE reduced GPx1 activity more significantly than did HNE or MG in a dose- and time-dependent manner. The addition of GSH to GPx1 3 h after incubation with ONE prevented further inhibition by trapping ONE as a ONE-GSH adduct. However, the activity of GPx1 was not restored to the initial level, indicating that ONE modified GPx1 irreversibly. This study suggests that oxidative damage to lipids, resulting in the formation of reactive aldehydes, can amplify cellular oxidative stress via direct inactivation of GPx1, which increases the production of intracellular peroxides.

Comparative studies for amyloid beta degradation: "Neprilysin vs insulysin", "monomeric vs aggregate", and "whole Aß40 vs its peptide fragments".

Amyloid beta (Aß) proteins are produced from amyloid precursor protein cleaved by ß- and γ-secretases, and are the main components of senile plaques pathologically found in Alzheimer's disease (AD) patient brains. Therefore, the relationship between AD and Aßs has been well studied for both therapeutic and diagnostic purposes. Several enzymes have been reported to degrade Aßs in vivo, with neprilysin (NEP) and insulysin (insulin-degrading enzyme, IDE) being the most prominent. In this article, we describe the mass spectrometric characterization of peptide fragments generated using NEP and IDE, and clarify the differences in digestion specificities between these two enzymes for non-aggregated Aß40, aggregated Aß40, and Aß40 peptide fragments, including Aß16. Our results allowed identification of all the peptide fragments from non-aggregated Aß40: NEP, 23 peptide fragments consisting of 2-11 amino-acid residues, 17 cleavage sites; IDE, 23 peptide fragments consisting of 6-33 amino-acid residues, 15 cleavage sites. Also, we confirmed that IDE can digest only whole Aß40, whereas NEP can digest both Aß40 and partial structures such as Aß16 and peptide fragments generated by the digestion of Aß40 by IDE. Furthermore, we confirmed that IDE and NEP are unable to digest aggregated Aß40.

Synthesis of deuterium-labeled analogs of the lipid hydroperoxide-derived bifunctional electrophile 4-oxo-2(E)-nonenal.

Lipid hydroperoxides undergo homolytic decomposition into the bifunctional 4-hydroxy-2(E)-nonenal and 4-oxo-2(E)-nonenal (ONE). These bifunctional electrophiles are highly reactive and can readily modify intracellular molecules including glutathione (GSH), deoxyribonucleic acid (DNA) and proteins. Lipid hydroperoxide-derived bifunctional electrophiles are thought to contribute to the pathogenesis of a number of diseases. ONE is an α,ß-unsaturated aldehyde that can react in multiple ways and with glutathione, proteins and DNA. Heavy isotope-labeled analogs of ONE are not readily available for conducting mechanistic studies or for use as internal standards in mass spectrometry (MS)-based assays. An efficient onestep cost-effective method has been developed for the preparation of C-9 deuterium-labeled ONE. In addition, a method for specific deuterium labeling of ONE at C-2, C-3 or both C-2 and C-3 has been developed. This latter method involved the selective reduction of an intermediate alkyne either by lithium aluminum hydride or lithium aluminum deuteride and quenching with water or deuterium oxide. The availability of these heavy isotope analogs will be useful as internal standards for quantitative studies employing MS and for conducting mechanistic studies of complex interactions between ONE and DNA bases as well as between ONE and proximal amino acid residues in peptides and proteins.