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Cells are the most basic structural and functional unit of living organisms. In recent years, people have paid more attention to cell heterogeneity. In order to characterize the heterogeneity of cells and prevent the life information in an individual cell from being overwhelmed by the average level of the population gained from traditional cell biology research, many new analytical techniques have been developed for single-cell analysis. However, single-cell analysis is extremely challenging due to the minimal cell volume, low content and wide variety of analytes in single cells, some of analytes change rapidly in cells and others may interfere with each other during analysis. Mass spectrometry has gradually become an ideal tool for single-cell analysis due to its high sensitivity, high resolution and high selectivity. In this review, several techniques of single-cell analysis based on mass spectrometry in the recent five years were systematically summarized and discussed including electrospray ionization mass spectrometry (ESI-MS), laser desorption ionization mass spectrometry (LDI-MS), secondary ion mass spectrometry (SIMS) and inductively coupled plasma mass spectrometry (ICP-MS). Finally, this review also looked into future developments of these mass spectrometry-based techniques. With the development of cell biology, the requirement for single-cell analysis technology is increasing as well. In the meanwhile, single-cell analysis, which focuses on studying the mechanisms of cellular and molecular behavior, has aroused more and more attention from various branches of life sciences in recent years. Although higher spatial resolution and detection sensitivity have become the development trends for single-cell mass spectrometry, none of these mass spectrometry-based techniques can be recognized flawless, which means they all have certain flaws to some extent. Consequently, single-cell analysis technology based on mass spectrometry is developing towards methodological diversification. On the one hand, it is of great importance to choose one suitable mass spectrometry-based technique according to the research purpose and actual conditions. On the other hand, it should be also emphasized that different methods can complement each other and jointly provide a more comprehensive analysis environment for single-cell mass spectrometry, thereby improving and innovating existing single-cell analysis techniques based on mass spectrometry. This review will provide insights into better understanding on and development of single-cell analysis by mass spectrometry.

Honey, renowned for its nutritional and therapeutic properties, has recently come under scrutiny due to its contamination by microplastics, in multiple ways. Bees' exposure to plastic pollution impacts the whole hive's ecosystem, and plastic tends to accumulate in hive products. Plastic packaging as polyethylene terephthalate (PET) is used to store honey in small flexible packages, which also increases the risk of microplastic migration. This study aims to establish three practical detection methods for PET microplastics and nanoplastics in honey, using readily available laboratory equipment without the need for chemical digestion or costly pretreatment protocols, in a laboratory-based simulation. The first method utilizes Raman micro-spectroscopy, offering high-resolution identification of PET microplastics on cellulose acetate filters with Raman mapping, eliminating the need for organic solvents or dyes. The second method employs optical microscopic observation under fluorescence with the aid of 4-dimethylamino-4′-nitrostilbene dye and ultraviolet radiation to enhance microplastic visibility, making it suitable for laboratories with standard optical microscopes. To isolate MPs from the solid honey particles, a density separator has been introduced using pentane. Lastly, the third method employs the use of electrospray ionization mass spectrometry for the detection of nanoplastics (<200 nm) in honey samples, through the examination of the different extraction phases of density separation. All the aforementioned methods contribute to efficient microplastic detection in honey, ensuring its quality and safe consumption. [ABSTRACT FROM AUTHOR]

Cells are the most basic structural and functional unit of living organisms. In recent years, people have paid more attention to cell heterogeneity. In order to characterize the heterogeneity of cells and prevent the life information in an individual cell from being overwhelmed by the average level of the population gained from traditional cell biology research, many new analytical techniques have been developed for single-cell analysis. However, single-cell analysis is extremely challenging due to the minimal cell volume, low content and wide variety of analytes in single cells, some of analytes change rapidly in cells and others may interfere with each other during analysis. Mass spectrometry has gradually become an ideal tool for single-cell analysis due to its high sensitivity, high resolution and high selectivity. In this review, several techniques of single-cell analysis based on mass spectrometry in the recent five years were systematically summarized and discussed including electrospray ionization mass spectrometry (ESI-MS), laser desorption ionization mass spectrometry (LDI-MS), secondary ion mass spectrometry (SIMS) and inductively coupled plasma mass spectrometry (ICP-MS). Finally, this review also looked into future developments of these mass spectrometry-based techniques. With the development of cell biology, the requirement for single-cell analysis technology is increasing as well. In the meanwhile, single-cell analysis, which focuses on studying the mechanisms of cellular and molecular behavior, has aroused more and more attention from various branches of life sciences in recent years. Although higher spatial resolution and detection sensitivity have become the development trends for single-cell mass spectrometry, none of these mass spectrometry-based techniques can be recognized flawless, which means they all have certain flaws to some extent. Consequently, single-cell analysis technology based on mass spectrometry is developing towards methodological diversification. On the one hand, it is of great importance to choose one suitable mass spectrometry-based technique according to the research purpose and actual conditions. On the other hand, it should be also emphasized that different methods can complement each other and jointly provide a more comprehensive analysis environment for single-cell mass spectrometry, thereby improving and innovating existing single-cell analysis techniques based on mass spectrometry. This review will provide insights into better understanding on and development of single-cell analysis by mass spectrometry. [ABSTRACT FROM AUTHOR]

Mass spectrometry has become an invaluable tool for real-time reaction monitoring and capturing highly reactive intermediates, significantly contributing to the understanding of various chemical reactions mechanisms. Recent studies involving the capture of elusive carbocations from different reactions within water microdroplets have drawn considerable attention. Inspired by George Olah's pioneering works on carbocations and superacid chemistry, they attributed the stabilization of transient carbocations to the acidic environment spontaneously formed on the surface of microdroplets, as per microdroplets' intriguing feature. In this work, the electrospray ionization mass spectrometry (ESI-MS) was used to determine a variety of carbocation precursor molecules mentioned. It's important to note that commercial ESI sources are typically unaffected by microdroplet effects due to the short lifespan of the droplets, resulting from an extremely short flight distance and immediate evaporation facilitated by the drying gas. Surprisingly, even in the absence of the ultra-acidic environment considered necessary for the stabilization of carbocations, the carbocation signals are able to be detected under specific analytical conditions. By studying the mass spectra of the same analytes when changing the inlet capillary temperature and switching tube lens voltage, it was revealed that the precursor species might undergo in-source fragmentation due to the combined effects of the inlet capillary's high temperature and the high tube lens voltage. Tube lenses, designed to improve the ion transport efficiency, were located at the entrance from the first to the second pumping stage, where the remaining pressure could be around 133 Pa. The voltage was applied here right after the ions flow out of the inlet capillary. Therefore, the temperature and tube lens voltage might interact with each other, and together with the remaining pressure in this rough vacuum region, the ions could potentially collide with gaseous molecules, leading to the production of carbocation fragments. These fragments, which were in consistent with those generated from collision-induced disassociation (CID) of the precursor species, could possibly be misinterpreted as intermediates of some certain reactions, thereby misleading our understanding of the reaction mechanism. Through the detailed investigation of instrument parameters, and a closer look at the structure of the atmospheric pressure interface was achieved, this work highlights the importance of carefully considering various instrument parameters when mass spectrometry is used for reaction monitoring and intermediate capture studies, to ensure the accuracy of the results, and avoid such pitfalls. [ABSTRACT FROM AUTHOR]

In recent years, top-down mass spectrometry has become a widely used approach to study proteoforms; however, improving sequence coverage remains an important goal. Here, two different proteins, α-synuclein and bovine carbonic anhydrase, were subjected to top-down collision-induced dissociation (CID) after electrospray ionisation. Two high-boiling solvents, DMSO and propylene carbonate, were added to the protein solution in low concentration (2%) and the effects on the top-down fragmentation patterns of the proteins were systematically investigated. Each sample was measured in triplicate, which revealed highly reproducible differences in the top-down CID fragmentation patterns in the presence of a solution additive, even if the same precursor charge state was isolated in the quadrupole of the instrument. Further investigation supports the solution condition-dependent selective formation of different protonation site isomers as the underlying cause of these differences. Higher sequence coverage was often observed in the presence of additives, and the benefits of this approach became even more evident when datasets from different solution conditions were combined, as increases up to 35% in cleavage coverage were obtained. Overall, this approach therefore represents a promising opportunity to increase top-down fragmentation efficiency., (© 2023 The Authors. Proteomics published by Wiley-VCH GmbH.)

A high-performance liquid chromatographic (HPLC) method coupled with diode-array detection (DAD) was optimized for the qualitative analysis of aqueous extracts of Cyclopia genistoides. Comprehensive insight into the phenolic profile of unfermented and fermented sample extracts was achieved with the identification of ten compounds based on comparison with authentic reference standards and the tentative identification of 30 additional compounds by means of electrospray ionization mass spectrometry (ESI-MS) and tandem MS detection. Three iriflophenone-di-O,C-hexoside isomers, three xanthone-dihydrochalcone derivatives and one dihydrochalcone are herein tentatively identified for the first time in C. genistoides. Of special interest is one iriflophenone-di-O,C-hexoside present in large amounts. New compounds (tentatively) identified for the first time in this species, and also in the genus Cyclopia, include two aromatic amino acids, one flavone, an iriflophenone-di-C-hexoside, a maclurin-di-O,C-hexoside, two tetrahydroxyxanthone-C-hexoside isomers, a tetrahydroxyxanthone-di-O,C-hexoside, two symmetric tetrahydroxyxanthone-C-hexoside dimers, nine glycosylated flavanone derivatives and five glycosylated phenolic acid derivatives. The presence of new compound subclasses in Cyclopia, namely aromatic amino acids and glycosylated phenolic acids, was demonstrated. The HPLC-DAD method was successfully validated and applied to the quantitative analysis of the paired sample extracts. In-depth analysis of the chemical composition of C. genistoides hot water extracts gave a better understanding of the chemistry of this species that will guide further research into its medicinal properties and potential uses.

Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein-protein and protein-ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as 'native MS,' has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure-function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by 'native MS,' when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions. [ABSTRACT FROM AUTHOR]

It is well-known that matrix effects in high performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) can seriously compromise quantitative analysis and affect method reproducibility. Paired ion electrospray ionization (PIESI) mass spectrometry is an approach for analyzing ultra-low levels of anions in the positive ion mode. This approach uses a structurally optimized ion pairing reagent to post-column associate with the anionic analyte, subsequently forming positively charged complexes. These newly formed complex ions are often more surface-active as compared to either the native anion or the ion pairing reagent. No studies have examined whether or not the PIESI approach mitigates matrix effects. Consequently, a controlled study was done using five analytes in highly controlled and reproducible synthetic groundwater and urine matrices. In addition, two different mass spectrometers (linear ion trap and triple quadrupole) were used. Compared to the negative ion mode, the PIESI-MS approach was less susceptible to matrix effects when performed on two different MS platforms. Using PIESI-MS, less dilution of the sample is needed to eliminate ionization suppression which, in turn, permits lower limits of detection and quantitation. [ABSTRACT FROM AUTHOR]

Corn peptides (CPs) are reported to have many biological functions, such as facilitating alcohol metabolism, antioxidation, antitumor, antihypertension, and hepatoprotection. To develop a method for quality control, the high-performance liquid chromatography (HPLC) system was applied. Twenty-eight common peaks were found in all the CPs of corn samples from Enshi, China, based on which, a fingerprinting chromatogram was established for use in quality control in future research. Subsequently, the major chemical constituents of these common peaks were identified respectively using the HPLC-diodearray detection electrospray ionization tandem mass spectrometry (DAD-ESI-MS/MS) system, and 48 peptide fractions were determined ultimately. This was the first time for the majority of these peptides to be reported, and many of them contained amino acids of glutamine (Q), L and A, which might play an important role in the exhibition of the bioactivities of CPs. Many peptides had a similar primary structure to the peptides which had been proven to be bioactive such as facilitating alcohol metabolism, scavenging free radicals, and inhibiting lipid peroxidation. This systematical analysis of the primary structure of CPs facilitated subsequent studies on the relationship between the structures and functions, and could accelerate holistic research on CPs. [ABSTRACT FROM AUTHOR]

The chemical components of navel orange juice were analyzed by wooden-tip electrospray ionizaion mass spectrometry (wooden-tip ESI-MS) without any sample pretreatment. The results show that signals of choline, proline, glucose and sucrose are rapidly obtained in positive ion detection mode. Among them, choline and proline give a signal of ［M+H］+, and glucose and sucrose produce a signal of ［M+K］+. While in negative ion detection mode, organic acids including fatty acid, phenolic acids and hydroxyl acids are readily detected. Meanwhile, wooden-tip ESI-MS is developed for semi-quantification determination of ascorbic acid in navel orange juice. The limit of detection (LOD) is 3.7 μg/L, the relative standard deviations (RSD) are 7.4%-9.7%, and the analytical time of each detection is less than 0.5 min. Wooden-tip ESI-MS has the advantages of minor or no sample pretreatment, convenient operation, sensitivity and high analytical speed, which will being an attractive method to rapidly screen the quality of agricultural products. [ABSTRACT FROM AUTHOR]

This paper describes the first study of the interactions of Ag+ with the organophosphorus (OP) insecticide diazinon, 1. Electrospray ionization mass spectrometry (ESI-MS) with corroborative collision-induced dissociation-mass spectrometry (CID-MS) demonstrates that 1 forms a bidentate chelate with Ag+. The hydrolysis products of 1, the pyrimidinol ( PY) and O, O-diethylphosphorothioic acid ( PA), are also found to bind to Ag+ via N ( PY) and S ( PA) Lewis base sites, respectively. 31P and 1H nuclear magnetic resonance (NMR) spectra in solution, followed over time with varying ratios of Ag+ to 1, confirm the MS evidence and show Ag+ catalysis of hydrolysis (e.g., complete hydrolysis of 1 in ∼5 min (first-order half-life = 3 × 10−4 d; kobs = 2 × 103 d−1)) with equimolar Ag+; this represents an approximate 150 000-fold enhancement in hydrolysis with Ag+ as compared to its absence. A mechanism for the enhanced hydrolysis is proposed in which bidentate binding of Ag+ to S of the P=S electrophilic site in tandem with binding to N of the leaving group stabilizes the SN2(P) transition state relative to the ground state; this effect is described by qualitative energy profiles. [ABSTRACT FROM AUTHOR]

Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance. However, quantification of DAG molecular species in various biological samples represents a challenging task because of their low abundance, hydrophobicity, and instability. This chapter describes the most common chromatographic (TLC and HPLC) and mass spectrometry (MS) methods used to analyze DAG molecular species. In addition, we directly compared the three methods using DAG obtained by phospholipase C hydrolysis of phosphatidylcholine purified from a Nicotiana benthamiana leaf extract. We conclude that each method identified similar major molecular species, however, the exact levels of those varied mainly due to sensitivity of the technique, differences in sample preparation, and processing. This chapter provides three different methods to analyze DAG molecular species, and the discussion of the benefits and challenges of each technique will aid in choosing the right method for your analysis., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The formation of molybdosilicate in the molybdate yellow method is elucidated by ESI-MS. Polymeric silicic acids were hydrolyzed to monomers, and molybdosilicates with a Keggin structure were formed. Each step of the formation reaction of molybdosilicate from molybdic and silicic acids between pH 1 and 2 was clarified by ESI-MS at the molecular level. In a solution below pH 0.5, the polymers of the molybdic acids were decomposed because of the high concentration of sulfuric acid. Excess sulfuric acid prevented the formation of molybdosilicate. Thus, none of the silicic acids reacted to form molybdosilicate at this pH, despite the presence of silicic acid and sulfate ions in the solution. Above pH 2, the condensation of molybdic acids occurred to form α-molybdosilicate. ESI-MS provides information on the formation of molybdosilicate and the individual reaction species present in the reaction mixture. [ABSTRACT FROM AUTHOR]

A high-performance liquid chromatographic (HPLC) method coupled with diode-array detection (DAD) was optimized for the qualitative analysis of aqueous extracts of Cyclopia genistoides. Comprehensive insight into the phenolic profile of unfermented and fermented sample extracts was achieved with the identification of ten compounds based on comparison with authentic reference standards and the tentative identification of 30 additional compounds by means of electrospray ionization mass spectrometry (ESI-MS) and tandem MS detection. Three iriflophenone-di-O,C-hexoside isomers, three xanthone-dihydrochalcone derivatives and one dihydrochalcone are herein tentatively identified for the first time in C. genistoides. Of special interest is one iriflophenone-di-O,C-hexoside present in large amounts. New compounds (tentatively) identified for the first time in this species, and also in the genus Cyclopia, include two aromatic amino acids, one flavone, an iriflophenone-di-C-hexoside, a maclurin-di-O,Chexoside, two tetrahydroxyxanthone-C-hexoside isomers, a tetrahydroxyxanthone-di-O,Chexoside, two symmetric tetrahydroxyxanthone-C-hexoside dimers, nine glycosylated flavanone derivatives and five glycosylated phenolic acid derivatives. The presence of new compound subclasses in Cyclopia, namely aromatic amino acids and glycosylated phenolic acids, was demonstrated. The HPLC-DAD method was successfully validated and applied to the quantitative analysis of the paired sample extracts. In-depth analysis of the chemical composition of C. genistoides hot water extracts gave a better understanding of the chemistry of this species that will guide further research into its medicinal properties and potential uses. [ABSTRACT FROM AUTHOR]

Abstract: The human islet polypeptide (hIAPP) or amylin is a 37-residue peptide hormone secreted by β-cells of the islet of Langerhans in the pancreas. Unlike the rat variant of IAPP (rIAPP), human amylin is highly amyloidogenic and is found as amyloid deposits in nearly 95% of patients afflicted with type 2 diabetes mellitus (T2DM). Human and rat IAPP have nearly identical primary sequence differing at only six positions which are encompassed within the 17–29 aminoacid region. Using Circular Dichroism (CD), Dynamic Light Scattering (DLS) and ThT-fluorescence (Th-T), we examined the aggregation properties of both full-length hIAPP1–37 and the related peptide fragment hIAPP17–29. For the sake of comparison, similar experiments were carried out on the respective rat variants rIAPP1–37 and rIAPP17–29. These studies were conducted at physiological pH in buffered solution not containing fluorinated co-solvents as well as in the presence of model membranes (LUV). In addition, the cytotoxic activity of the investigated peptides was determined toward different pancreatic β-cell lines. All the peptide studied in this work resulted cytotoxic despite β-sheet structure being observed, in vitro, for the hIAPP1–37 only. This suggests that β-sheet conformational transition that generally precedes the fibril formation, is not a prerequisite for toxicity towards β-cells. Interestingly, confocal microscopy indicated that the IAPP peptides can enter the cell and might exert their toxic action at an intracellular level. [Copyright &y& Elsevier]

Highlights: [•] α-Linolenic acid (ALA) was biotransformed by Aspergillus niger into species with different degree of oxygenation. [•] The main biotransformation products were characterized by LC-MS/MS analyses. [•] Their anti-inflammatory potential was evaluated with NO and TNF-α quantification in LPS-stimulated RAW264.7 macrophages. [•] The isomeric ketoacids 9-KOTE and 13-KOTE were the most active. [•] Biotransformation was successfully employed for the production of potential analogs to lipid mediators. [Copyright &y& Elsevier]

Abstract: A series of 4-(4-substituted piperazin)-5,6,7-trialkoxy quinazoline was prepared by conventional heating methods. Among these compounds, the crystal structure of compound 10o (CCDC: 916922) was determined by X-ray crystallography. Bioassay results showed that most target compounds had certain inhibition activities against proliferation of tumor cells, and some compounds even had good broad-spectrum inhibition activities. The ethoxyl series of compounds possessed higher inhibition activities against tumor cells than the methoxyl series of compounds. Bioactivity tests showed that the IC50 values of compound 10s against PC3, MGC803, A375, and A549 cells were 1.8, 2.8, 1.3, and 2.9 μΜ, respectively, which were much higher than those of commercial gefitinib (7.2, 7.6, 7.2, and 9.8 μM, respectively). Conversely, the IC50 values of compound 10s were very low against NH3T3, indicating only weak effect on normal cells as also proven by lactate dehydrogenase and acridine orange/ethidium bromide staining. Analyses of cell configuration and cell cycle revealed that compound 10s possibly caused cells to remain at G0/G1 phase by inhibiting cell proliferation for 24 h. Compound 10s also inhibited the phosphorylation of ERK1/2 and P38 with obvious concentration dependence. Thus, these compounds can inhibit the proliferation of A549 cells through the interruption of ERK1/2 and P38signaling pathways. [Copyright &y& Elsevier]

Abstract: The aggregation of the microtubule-associated protein tau into paired helical filaments to form neurofibrillary tangles constitutes one of the pathological hallmarks of Alzheimer's disease. Tau is post-translationally modified by the addition of N-acetyl-d-glucosamine O-linked to several serine and threonine residues (O-GlcNAc). Previously, increased O-GlcNAcylation of tau has been shown to block the accumulation of tau aggregates within a tauopathy mouse model. Here we show that O-GlcNAc modification of full-length human tau impairs the rate and extent of its heparin-induced aggregation without perturbing its activity toward microtubule polymerization. O-GlcNAcylation, however, does not impact the “global-fold” of tau as measured by a Förster resonance energy transfer assay. Similarly, nuclear magnetic resonance studies demonstrated that O-GlcNAcylation only minimally perturbs the local structural and dynamic features of a tau fragment (residues 353–408) spanning the last microtubule binding repeat to the major GlcNAc-acceptor Ser400. These data indicate that the inhibitory effects of O-GlcNAc on tau aggregation may result from enhanced monomer solubility or the destabilization of fibrils or soluble aggregates, rather than by altering the conformational properties of the monomeric protein. This work further underscores the potential of targeting the O-GlcNAc pathway for potential Alzheimer's disease therapeutics. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: The root bark of Lycium chinense Miller, Lycii Radicis Cortex, has been used in traditional Chinese medicine (TCM) to treat different inflammation-related symptoms, such as diabetes mellitus. The pro-inflammatory transcription factor nuclear factor kappa B (NF-?B) is a key regulator of inflammation, while the transcription factor peroxisome proliferator-activated receptor gamma (PPAR?) is a key modulator of genes involved in diabetes development. To identify putative active compound(s) from Lycii Radicis Cortex inhibiting NF-?B or activating PPAR?. Material and methods: Using activity-guided fractionation, six extracts with different polarity, isolated fractions, and purified compounds from Lycii Radicis Cortex were tested for NF-?B inhibition and PPAR? activation in vitro. The structure of the purified compounds was elucidated by NMR and MS techniques. Results: The ethyl acetate extract and the methanol extract of Lycii Radicis Cortex suppressed tumor necrosis factor alpha (TNF-?)-induced activation of NF-?B, while the dichloromethane extract activated PPAR?. Nine phenolic amide analogues, including trans-N-(p-coumaroyl)tyramine (1), trans-N-feruloyltyramine (2), trans-N-caffeoyltyramine (3), dihydro-N-caffeoyltyramine (4), three neolignanamides (5–7), and two lignanamide (8, 9), were isolated and their inhibitory potential on NF-?B was determined (1–4 were also contained in water decoction). Two of the nine isolated phenolic amides inhibited TNF-?-induced NF-?B activation. Trans-N-caffeoyltyramine was verified as the key component responsible for the NF-?B inhibition with an IC50 of 18.4?M in our cell-based test system. Activation of PPAR? was attributed to a palmitic-acid enriched fraction which displayed concentration-dependent effect ablated upon co-treatment with the PPAR? antagonist T0070907. Conclusions: Phenolic amides were confirmed as main components from Lycii Radicis Cortex responsible for NF-?B inhibition. Fatty acids were identified as the major plant constituent responsible for the PPAR? activation. Structure-activity relationship analysis suggests that the NF-?B inhibitory activity of trans-N-caffeoyltyramine may be attributed to its Michael acceptor-type structure (?,?-unsaturated carbonyl group). The data of this study contribute to a better understanding of the molecular mechanism of action of Lycii Radicis Cortex extracts in the context of inflammation. [Copyright &y& Elsevier]

Abstract: We previously reported the identification and development of novel inhibitors of streptokinase (SK) expression by Group A Streptococcus (GAS), originating from a high throughput cell-based phenotypic screen. Although phenotypic screening is well-suited to identifying compounds that exert desired biological effects in potentially novel ways, it requires follow-up experiments to determine the macromolecular target(s) of active compounds. We therefore designed and synthesized several classes of chemical probes for target identification studies, guided by previously established structure–activity relationships. The probes were designed to first irreversibly photolabel target proteins in the intact bacteria, followed by cell lysis and click ligation with fluorescent tags to allow for visualization on SDS–PAGE gels. This stepwise, ‘tag-free’ approach allows for a significant reduction in molecular weight and polar surface area compared to full-length fluorescent or biotinylated probes, potentially enhancing membrane permeability and the maintenance of activity. Of the seven probes produced, the three most biologically active were employed in preliminary target identification trials. Despite the potent activity of these probes, specific labeling events were not conclusively observed due to a considerable degree of nonspecific protein binding. Nevertheless, the successful synthesis of potent biologically active probe molecules will serve as a starting point for initiating more sensitive methods of probe-based target identification. [Copyright &y& Elsevier]

Highlights: [•] CSPC inhibits HepG2 cell proliferation in a dose-dependent manner (100–400μg/mL). [•] HepG2 cell cycle is arrested by CSPC in the G0/G1 phase. [•] CSPC can induce apoptosis and trigger necrosis of HepG2 cell. [•] CSPC causes a loss of mitochondrial membrane potential in HepG2 cell. [•] CSPC stimulates reactive oxidative species generation in HepG2 cell. [Copyright &y& Elsevier]

Abstract: Background: Proteins are extremely reactive to oxidants and should represent a potential target of instable reactive oxygen. This may represent a problem for plasma proteins since they may be directly modified in vivo in a compartment where antioxidant enzymatic systems are scarcely represented. On the other hand, it is possible that some plasma components have evolved over time to guarantee protection, in which case they can be considered as anti-oxidants. Scope of review: To present and discuss main studies which addressed the role of albumin in plasma antioxidant activity mainly utilizing in vitro models of oxidation. To present some advances on structural features of oxidized albumin deriving from studies carried out on in vitro models as well as albumin purified in vivo from patients affected by clinical conditions characterized by oxidative stress. Major conclusions: There are different interaction with HOCl and chloramines. In the former case, HOCl produces an extensive alteration of 238Trp and 162Tyr, 425Tyr, 47Tyr, while thiol groups are only partially involved. Chloramines are extremely reactive with the unique free SH group of albumin (34Cys) with the formation of sulfenic and sulfinic acid as intermediates and sulfonic acid as end-product. Oxidized albumin has a modified electrical charge for the addition of an acidic residue and presents α-helix and random coil reorganization with subtle changes in domain orientation. General significance: Albumin, is the major antioxidants in plasma with a concentration (0.8mM) higher than other antioxidants by an exponential factor. Functional and protective roles in the presence of oxidative stress must be defined. This article is part of a Special Issue entitled Serum Albumin. [Copyright &y& Elsevier]

Abstract: The study of noncovalent interactions between pharmaceutical molecules and proteins is essential for understanding molecular mechanisms of protein function, and provides foundations for de novo therapeutic agent design. Electrospray ionization mass spectrometry (ESI-MS) has nowadays become a popular tool for analyzing the noncovalent protein complexes, however it usually has difficulty in determining the interaction sites and binding mechanisms. In this work, a new structure-differential binding (SDB) method, combined with ESI-MS and molecular docking (MD) techniques (SDB–ESIMS–MD), was developed and applied to a study of the binding interactions in noncovalent protein–small drug molecule complexes for the characterization of binding sites and binding modes. Using this developed method, protein complexes of flavonoid and flavonoid glycoside ligands and cytochrome-c (Cyt-c) were studied in detail. ESI-MS was used to determine the relative binding affinities and dissociation constants of flavonoid–Cyt-c complexes, and to measure the changes in the stability of the protein complexes with the structural modifications of the ligands for identifying effective binding functional groups. Molecular docking simulations complemented ESI-MS experiments by providing the protein–ligand interaction profile of each complex and displaying the binding mode for each interaction. This SDB–ESIMS–MD method can be applied to a broad range of protein–drug interactions and used to guide further research in the study of structure–binding relationship between drug molecules and targeted biomacromolecules. [Copyright &y& Elsevier]

Abstract: Bombesin is a neuropeptide widely studied due to its ability to target various types of cancers. Technetium-99m on the other hand is ideal for diagnostic tumor targeting. The aim of the present study is the investigation of the coupling of the ligand (S)-(2-(2′-pyridyl)ethyl)-d,l-cysteine with the BN-peptide Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met(CONH2) through the spacer aminohexanoic acidand the labeling of the resulting derivative MBN with the synthon [M(CO)3(H2O)3]+ (M= 99mTc, Re). The peptide was synthesized according to the SPPS method, purified and characterized by ESI-MS. The new 99mTc-labeled biomolecule was stable in vitro, showed high affinity for the human GRP receptor expressed in PC3 cells and the rate of internalization was found to be time-dependent tissue distribution of the radiopeptide was evaluated in normal mice and in prostate cancer experimental models and significant radioactivity uptake was observed in the pancreas of normal mice as well as in PC3 tumors. Dynamic studies of the radiopeptide showed satisfactory tumor images. [Copyright &y& Elsevier]

Abstract: Metallothioneins (MTs) are small cysteine-rich proteins with the ability to coordinate heavy metal atoms through metal–thiolate bonds, which are widely distributed among the animal and plant kingdoms. Multifunctional roles for MTs have been proposed, including their ability to scavenger various radicals and reactive oxygen species. In the present article we summarize available information of four MT polypeptides from different organisms, forming metal complexes with Zn(II), Cd(II) or Cu (I) ions. Non-enzymatic modifications of MTs under ionizing radiations and their consequences on the lipidic membrane compartment were studied by Raman spectroscopy and a biomimetic model, respectively. The latter is based on liposome technology and allows to measure the trans unsaturated fatty acid content as a result of reductive radical stress on MTs. Biological significance: The effect of radical stress on the cell metabolism and functions is a very active field of research connecting various disciplines in life sciences. In this contest tandem radical damage has been the subject of recent investigations that pointed out its harmfulness in the general scenario of establishing the consequences of radical stress. By using biomimetic models of tandem damage we have for the first time tested the capability of metallothioneins (MTs), small metalloproteins rich of Cys residues, to damage another cell compartment like lipid membranes when they are undergone to reductive radical stress. The connection of MT reactivity with membrane lipid transformation can give a contribution to the puzzling context of radical stress occurring to biomolecules and the role as biological signaling. To this purpose, MT polypeptides from different organisms, exhibiting different sequence peculiarities, have been analyzed here. The spectroscopic analysis of these systems has allowed to identify modifications affecting metal–thiolate clusters, cystines, and Met residues, acting as efficient interceptors of reducing radical species. The chemical mechanism involving sulfur-containing moieties under reductive conditions discloses new scenarios that bring to the loss of sulfur-centered radicals by desulfurization reactions that change the natural sequences of MTs. Ala is a genetically coded amino acid, therefore the mutation of Cys to Ala occurring to a sequence by the radical process so far discussed, corresponds to a post-translational modification. Research on such mutation connected also to a free radical stress will be important to contribute for a complete picture of the degeneration associated to diseases and aging. Analogously, the Met to Aba mutation occurring after reductive stress transforms a natural amino acid into a natural, non-genetically-coded congener. Aba corresponds to a homologation of the alkyl chains normally present in genetically codified amino acids, such as methyl (in Ala) and isopropyl (in Leu), with an ethyl unit. Based on alkyl substitution, this modification can therefore be studied in order to understand its general consequences on the structure–activity relationships in proteins and, in particular, on molecular interactions. This article is part of a Special issue entitled: Posttranslational Protein modifications in biology and Medicine. [Copyright &y& Elsevier]

Abstract: Arsenic is an element widely present in nature. Additionally, it may be found as different species in several matrices and therefore it is one of the target elements in chemical speciation. Although the number of studies in terrestrial plants is low, compared to matrices such as fish or urine, this number is raising due to the fact that this type of matrix are closely related to the human food chain. In speciation analysis, sample preparation is a critical step and several extraction procedures present drawbacks. In this review, papers dealing with extraction procedures, analytical methods, and studies of species conservation in plants cultivated in terrestrial environment are critically discussed. Analytical procedures based on extractions using water or diluted acid solutions associated with HPLC–ICP–MS are good alternatives, owing to their versatility and sensitivity, even though less expensive strategies are shown as feasible choices. [Copyright &y& Elsevier]

Highlights: [•] Cyclometalated complexes of noble metals are promising anticancer prodrugs. [•] These compounds show improved stability and enhanced cytotoxicity. [•] Cyclometalated compounds are suitable for rational drug design. [•] Besides DNA, protein/enzyme interactions represent a major mode of action. [Copyright &y& Elsevier]

Highlights: [•] We determined the LPS structures of non-typeable H. influenzae strain 1232. [•] The strain expresses d,d-Hep in the outer core of LPS in a novel manner. [•] The branching at d,d-Hep was indicated by MS4 experiments and confirmed by NMR. [•] d,d-Hep is substituted at both O-4 and O-7. [ABSTRACT FROM AUTHOR]

A series of 5,6,7-trimethoxy-N-phenyl(ethyl)-4-aminoquinazoline compounds was prepared by microwave irradiation and conventional heating methods. Compounds 6p, 6q, and 6x strongly inhibited extracellular regulated kinase1/2 (ERK1/2) phosphorylation induced by epidermal growth factor (EGF) at 1.28 μM in PC3 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that all compounds had certain anticancer activities, and the IC50 values of 6x were 6.2 ± 0.9, 3.2 ± 0.1, and 3.1 ± 0.1 μM against PC3, BGC823, and Bcap37 cells, respectively. Acridine orange/ethidium bromide staining, Hoechst 33258 staining, DNA ladder, and flow cytometry analyses revealed that 6x induced cell apoptosis in PC3 cells, with apoptosis ratios of 11.6% at 1 μM and 31.8% at 10 μM after 72 h. [ABSTRACT FROM AUTHOR]

Milked venoms of Conus demonstrate direct lineage to US Food and Drug Administration approved and present in-trial drug leads. Yet the complexity of the milked venom has not been adequately investigated or characterized, in a sustainable manner. In this study we determine the extent of molecular mass differentiation in milked venom from captive Conus magus and confirm the expression of known conotoxin constituents. We demonstrate the presence of post-translational N-terminal peptide truncation, which differentiates the milked venom constituent α-conotoxin MI from the novel α-conotoxin MIC. This truncation has a direct effect on peptide bioactivity – K i of 89.1 ± 9.1 and 248.7 ± 10.9 nM (α-conotoxin MI and MIC respectively) toward the muscle-type nAChR (Torpedo). These milked venom conotoxins demonstrated acute lethality in fish, with a LD50 of 12.24 and 23.29 μg kg−1 for α-conotoxin MI and MIC respectively. By synthesizing and investigating the synthetic intermediate variant des[Gly]1α-conotoxin MI, it was demonstrated that retention of the N-terminal arginine residue increased affinity at the muscle-type nAChR site (binding K i of 73.3 ± 5.8 nM and lethal toxicity level LD50 of 8.19 μg kg−1). This post-translational modification event within the milked venom of C. magus represents a unique mechanism by which cone snails are able to increase the chemical and pharmacological diversity of their venoms. [ABSTRACT FROM AUTHOR]

Abstract: Viral suppressors of RNA interference (VSRs) target host gene silencing pathways, thereby operating important roles in the viral cycle and in host cells, in which they counteract host innate immune responses. However, the molecular mechanisms of VSRs are poorly understood. We provide here biochemical and biophysical features of the dual suppressor/activator VSR P1 protein encoded by the rice yellow mottle virus. In silico analyses of P1 suggested common features with zinc finger proteins and native mass spectrometry unambiguously confirmed that recombinant P1 binds reversibly two zinc atoms, each with a different strength. Additionally, we demonstrate that the reaction of P1 with H2O2 leads to zinc release, disulfide bond formation, and protein oligomerization. A reversible protein modification by redox alterations has only been described for a limited number of zinc finger proteins and has never been reported for VSRs. Those reported here for P1 might be a general feature of Cys-rich VSRs and could be a key regulatory mechanism for the control of RNA silencing. [Copyright &y& Elsevier]

Highlights: [•] Comprehensive N-glycosylation analysis techniques for biotherapeutics are described. [•] Identification of potentially immunogenic epitopes is very important. [•] Sample-preparation issues are discussed, including glycan release fluorophore labeling, sample cleanup and capillary electrophoresis analysis. [•] Carbohydrate sequencing reveals linkage and positional information. [•] Glycan analysis needs are different in clone selection, product development and lot release. [ABSTRACT FROM AUTHOR]

Highlights: [•] Intrafamily differences are found for C1 GH11 endo-xylanases. [•] GH11 endo-xylanases are influenced by solubility and molecular structure of xylan. [•] Valine instead of tyrosine at position 163 causes GH10-like degradation products. [•] GH11 xylanases release many small degradation products from self-associated xylans. [Copyright &y& Elsevier]

Background: The lack of analytical methods specific for each lipid class, particularly for phospholipids and sphyngolipids, makes necessary their separation by preparative techniques before quantification. LC–MS would be the election method but for daily work in the clinical laboratory this is not feasible for different reasons, both economic and time consuming. In the present work, we have optimized an HPLC method to quantify lipid classes in plasma and erythrocytes and applied it to samples from patients with cirrhosis. Methods: Lipid classes were analyzed by normal phase liquid chromatography with evaporative light scattering detection. We employed a quaternary solvent system to separate twelve lipid classes in 15min. Results: Interday, intraday and recovery for quantification of lipid classes in plasma were excellent with our methodology. The total plasma lipid content of cirrhotic patients vs control subjects was decreased with diminished CE (81±33 vs 160±17mg/dL) and PC (37±16 vs 60±19mg/dL). The composition of erythrocytes showed a decrease in acidic phospholipids: PE, PI and PS. Conclusion: Present methodology provides a reliable quantification of lipid classes in blood. The lipid profile of cirrhotics showed alterations in the PC/PE plasma ratio and in the phospholipid content of erythrocytes, which might reflect alterations in hepatocyte and erythrocyte membrane integrity. [ABSTRACT FROM AUTHOR]

The understanding of the function of macromolecular complexes is mainly related to a precise knowledge of their structure. Recently, the development of suitable mass spectrometric techniques (electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI)) and multi-angle laser light scattering has enabled mass determination of native complexes and of their subunits. By these techniques, the structure and association/dissociation behavior of huge molecules of molluscan Octopus vulgaris, Sepia officinalis and Rapana venosa have been characterized. Molecular masses of the native and dissociated molecule of cephalopodan Hcs O. vulgaris (3545 and 359.3 kDa, respectively) and S. officinalis (4134 and 443.8 kDa, respectively) revealed that only one type subunit organizes their molecules, while the presence of two isoforms with different masses (422.8 and 400.0 kDa) has been determined for gastropodan R. venosa Hc, aggregated into didecamers. The difference of their structural subunits was also established after limited proteolysis with TPCK-trypsin. Eight functional units (FUs) with masses of ~ 50 kDa were isolated from both subunits of RvH and isoform of Sepia officinalis, while seven FUs were purified from OvH. Further characterization of proteins by ESI-mass spectrometry (MS) and MALDI-MS, methods gave insights into post-translational modifications such as glycosylation. Glycosylation of O. vulgaris and S. officinalis Hcs was suggested based on the differences (11.6 and 40.0 kDa, respectively) between the masses measured by ESI-MS and those calculated by their gene sequences. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract: This report focuses on studies of lipid peroxidation products reactivity towards the side chains of cysteine, histidine, and lysine residues in structurally unordered peptides. Thus we have analyzed linoleic acid peroxidation products (LaPP) obtained by incubating 1-palmitoyl-2-linoleoyl-sn-glycerophosphatidylcholine (PLPC) overnight with or without H2O2 in the presence or absence of CuCl. In total, 55 different LaPP were identified with 26 containing reactive carbonyl groups. The strongest oxidation conditions (H2O2 and Cu(I), i.e. a Fenton-like reagent) yielded 51 LaPP, whereas air oxidation produced only 12 LaPP. Independent of the oxidation conditions, around half of all LaPP were short-chain (oxidative cleavage) and the others long-chain (oxygen addition) PLPC oxidation products. The stronger oxidation conditions increased the number of LaPP, but also oxidized the added peptide Ac-PAAPAAPAPAEXTPV-OH (X=Cys, His or Lys) very quickly, especially under Fenton conditions. Thus, PLPC was oxidized by milder conditions (air or Cu(I)), incubated with the peptide and the peptide modifications were then analyzed by nano-RPC-ESI-Orbitrap-MS. Ten LaPP-derived peptide modifications were identified at lysine, whereas nine products were identified for cysteine and only three for histidine. Three high molecular weight LaPP still esterified to the GPC backbone were detected on Lys-containing peptide. Furthermore, three LaPP-derived mass shifts were obtained at cysteine, which have not previously been reported. [Copyright &y& Elsevier]

Abstract: In the past decade, cell free DNA, or circulating cell free DNA, or cell free circulating DNA, isolated from body fluids such as plasma/serum/urine has emerged as an important tool for clinical diagnostics. The molecular biology of circulating cell free DNA is poorly understood but there is currently an increased effort to understand the origin, mechanism of its circulation, and sensitive characterization for the development of diagnostic applications. There has been considerable progress towards these goals using real-time polymerase chain reaction (rt-PCR) technique. More recently, new attempts to incorporate mass spectrometric techniques to develop accurate and highly sensitive high-throughput clinical diagnostic tests have been reported. This review focuses on the methods to isolate circulating cell free DNA from body fluids, their quantitative analysis and mass spectrometry based characterization in evolving applications as prenatal and cancer diagnostic tools. [Copyright &y& Elsevier]

Abstract: In order to study the different effects of ginsenosides with similar structures, research on interactions between ginsenoside Rg1, Re and lysozyme was carried out by electrospray ionization mass spectrometry (ESI-MS) and molecular docking. The 1:1 and 2:1 noncovalent complexes of ginsenosides and lysozyme were observed in the mass spectra and the dissociation constants for them were directly calculated based on peak intensities of lysozyme and its noncovalent complexes with ginsenosides. The results showed that the 1:1 complex of ginsenoside Rg1 and lysozyme was more stable than that of ginsenoside Re and lysozyme. As the acidity increased, the stabilities of the 1:1 complexes of Rg1, Re and lysozyme both decreased. Interestingly, as the acidity increased, the stability of the 2:1 complex of Rg1 and lysozyme increased while that of Re decreased. From the result of molecular docking, ginsenosides interacted with the active sites of lysozyme. And the stability of the complexes could be affected by the conformation changes of lysozyme as acidity increased. [ABSTRACT FROM AUTHOR]

Abstract: A reagent, 1-(4-isopropyl) phenyl-3-methyl-5-pyrazolone (PPMP) has been synthesized and used for high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) determination of pre-column labeled carbohydrates. Monosaccharides have been quantitatively converted into mono-PPMP-labeled derivatives with 28% aqueous ammonia as a catalyst at 80°C during 70min. Mono-PPMP derivatives have been demonstrated to exhibit better chemical stability than bis-PMP ones. PPMP-labeled mixture of twelve monosaccharides (galactosamine, glucosamine, galacturonic acid, glucuronic acid, galactose, glucose/N-acetylgalactosamine, N-acetylglucosamine, xylose, arabinose, mannose, fucose, and rhamnose) has been well separated by a reverse-phase HPLC and detected by on-line ESI-MS method under optimized conditions. The data on characteristic fragment ions of the 13 PPMP-labeled monosaccharides with MS2 data have been collected. The suggested method exhibits good linearity (correlation coefficients>0.9975) between the peak areas and the concentration of monosaccharides in a broad concentration range and good reproducibility (RSD<3.19%). The developed method has been successfully applied to analyze the monosaccharide composition of natural Spirulina polysaccharide SPPB-1. [Copyright &y& Elsevier]

In this work, we report our findings on the use of radical thiol‐ene chemistry for polymer–polymer conjugation. The manuscript combines the results from the Preparative Macromolecular Chemistry group from the Karlsruhe Institute of Technology (KIT) and the Polymer Chemistry Research group from Ghent University (UGent), which allowed for an investigation over a very broad range of reaction conditions. In particular, thermal and UV initiation methods for the radical thiol‐ene process were compared. In the KIT group, the process was studied as a tool for the synthesis of star polymers by coupling multifunctional thiol core molecules with poly(n‐butyl acrylate) macromonomers (MM), employing thermally decomposing initiators. The product purity and thus reaction efficiency was assessed via electrospray ionization mass spectrometry. Although the reactions with 10 or 5 equivalents of thiol with respect to macromonomer were successful, the coupling reaction with a one‐to‐one ratio of MM to thiol yielded only a fraction of the targeted product, besides a number of side products. A systematic parameter study such as a variation of the concentration and nature of the initiator and the influence of thiol‐to‐ene ratio was carried out. Further experiments with poly(styrene) and poly(isobornyl acrylate) containing a vinylic end group confirmed that thermal thiol‐ene conjugation is far from quantitative in terms of achieving macromolecular star formation. In parallel, the UGent group has been focusing on photo‐initiated thiol‐ene chemistry for the synthesis of functional polymers on one hand and block copolymers consisting of poly(styrene) (PS) and poly(vinyl acetate) (PVAc) on the other hand. Various functionalization reactions showed an overall efficient thiol‐ene process for conjugation reactions of polymers with low molecular weight compounds (∼90% coupling yield). However, while SEC and FT‐IR analysis of the conjugated PS‐PVAc products indicated qualitative evidence for a successful polymer–polymer conjugation, 1H NMR and elemental analysis revealed a low conjugation efficiency of about 23% for a thiol‐to‐ene ratio equal to one. Blank reactions using typical thiol‐ene conditions indicated that bimolecular termination reactions occur as competitive side reactions explaining why a molecular weight increase is observed even though the thiol‐ene reaction was not successful. The extensive study of both research groups indicates that radical thiol‐ene chemistry should not be proposed as a straightforward conjugation tool for polymer–polymer conjugation reactions. Head‐to‐head coupling is a major reaction pathway, which interrupts the propagation cycle of the thiol‐ene process. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1699–1713, 2010 [ABSTRACT FROM AUTHOR]

Hemoglobinopathies are the most frequent genetic diseases in the world. Among them, the Hb S variant [β6(A3)Glu→Val], which, in the homozygous state, produces a severe disease known as sickle cell anemia with polymerization of Hb S inside red blood cells under hypoxic conditions. Additional mutations, in cis or in trans of the βS-globin chain, may inhibit or enhance the polymerization process. We describe here a new hemoglobin (Hb) variant (Hb S-Clichy) which carries the βS-globin chain and an additional mutation β8(A5)Lys→Thr. The variant was detected by routine electrophoretic techniques and cation exchange liquid chromatography (CE-LC). Globin chain separation by reversed phase LC (RP-LC) showed normal and abnormal β chains, confirming that the additional abnormality was located in cis to Hb S. Electrospray ionization mass spectrometry (ESI-MS) gave a 57 Da mass decrease for the abnormal globin chain. The abnormal chain was isolated and submitted to trypsin digestion. Normal peptides βT-1 and βT-2 were not observed on the matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrum but a new peptide βT-1,2 was detected. Nano LC-ESI-MS/MS of the new peptide showed that the glutamic acid at codon 6 was replaced by a valine residue, and the lysine at codon 8 was replaced by a threonine residue, as confirmed by DNA sequencing. This example demonstrates that in a population where Hb S is present, every unidentified Hb needs to be clearly characterized to prevent major sickle cell syndromes. In addition, the identification of these variants must be considered in newborn screening for sickle cell disease, using either classical biochemical methods or MS techniques. [ABSTRACT FROM AUTHOR]

Lantibiotics are a group of potent antibacterial agents that contain unusual amino acids, such as the thioether amino acids lanthionine and methyllanthionine, and the didehydroamino acids didehydroalanine and didehydro-aminobutyric acid. Here, we report on an antibacterial lantibiotic peptide named SWLP1 ( Staphylococcus warneri lantibiotic peptide 1), which is secreted from Staphylococcus warneri (deposited with DSMZ, accession number DSM 16081). SWLP1 was purified from growth media. The purified peptide displays antibacterial activity against several species, including Staphylococcus epidermidis. The molecular mass of SWLP1 is 2998.9 Da as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The sequence and possible structure was elucidated by combining electrospray ionization mass spectrometry/mass spectrometry data of ethanethiol-treated and non-ethanethiol-treated tryptic fragments of the SWLP1. SWLP1 contains three thioether bridges, one didehydroalanine, and three didehydroaminobutyric acids. This peptide has the potential to be used in treatment of several Gram-positive bacterial infections. [ABSTRACT FROM AUTHOR]

Abstract: The anti-inflammatory selenium compounds, ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) and selenite, were found to alter the DNA binding mechanisms and structures of cysteine-rich zinc-finger transcription factors. As assayed by DNase I protection, DNA binding by TFIIIA (transcription factor IIIA, prototypical Cys2His2 zinc finger protein), was inhibited by micromolar amounts of ebselen. In a gel shift assay, ebselen inhibited the Cys2His2 zinc finger-containing DNA binding domain (DBD) of the NF-κB mediated transcription factor Sp1. Ebselen also inhibited DNA binding by the p50 subunit of the pro-inflammatory Cys-containing NF-κB transcription factor. Electrospray ionization mass spectrometry (ESI-MS) was utilized to elucidate mechanisms of chemical interaction between ebselen and a zinc-bound Cys2His2 zinc finger polypeptide modeled after the third finger of Sp1 (Sp1-3). Exposing Sp1-3 to micromolar amounts of ebselen resulted in Zn2+ release from this peptide and the formation of a disulfide bond by oxidation of zinc finger SH groups, the likely mechanism for DNA binding inhibition. Selenite was shown by ESI-MS to also eject zinc from Sp1-3 as well as induce disulfide bond formation through SH oxidation. The selenite-dependent inhibition/oxidation mechanism differed from that of ebselen by inducing the formation of a stable selenotrisulfide bond. Selenite-induced selenotrisulfide formation was dependent upon the structure of the Cys2His2 zinc finger as alteration in the finger structure enhanced this reaction as well as selenite-dependent zinc release. Ebselen and selenite-dependent inhibition/oxidation of Cys-rich zinc finger proteins, with concomitant release of zinc and finger structural changes, points to mechanisms at the atomic and protein level for selenium-induced alterations in Cys-rich proteins, and possible amelioration of certain inflammatory, neurodegenerative, and oncogenic responses. [Copyright &y& Elsevier]

When analyzed by routine cation exchange high performance liquid chromatography (HPLC), the Hb A1c peak of a Caucasian diabetic patient from Yorkshire, UK, appeared to be an incompletely resolved doublet. One component (5.5%) eluted at the normal time for Hb A1c, whereas the other component (6.6%) eluted slightly later. The HPLC trace was otherwise normal. Analysis of the diabetic patient's blood and a tryptic digest thereof by electrospray ionization mass spectrometry (ESI-MS) identified the Hb Belleville trait. To relate Hb A1c determined by HPLC to α and β chain glycation determined by ESI-MS, several normal blood samples (5-14% Hb A1c) were analyzed by both techniques. The Hb A1c levels derived from the α and β chain glycation levels of the diabetic patient (12.9 and 12.4%, respectively) agreed with the sum of the two peaks (12.1%) in the HPLC trace. Similarly, Hb Belleville was detected and identified in another Caucasian, also from Yorkshire, with normal Hb A1c. [ABSTRACT FROM AUTHOR]

Electrospray ionization mass spectrometry (ESI-MS) data and molecular modeling calculations were used to gain mechanistic, conformational, and domain-specific information from the acid-induced demetallation reactions of human metallothionein. The recombinant proteins studied were the single α- and β-rhMT-1a domains and the βα- and αβ-rhMT-1a two-domain species, based on the human metallothionein 1a sequence. Complete molecular models (MM3/MD) for all the fully metallated and demetallated species using a modified force field are reported for the first time. Basic residues that contribute to the ESI-MS charge states are identified from the molecular models. Demetallation took place under equilibrium conditions within a narrow pH range. For the two-domain proteins, these results support a demetallation mechanism involving the initial complete demetallation of one domain followed by the other for both βα-rhMT and αβ-rhMT. Based on the stability of the separate domains, the β domain is predicted to demetallate first in the two-domain rhMTs. Both the α domain and the β domain were observed to bind an excess of one Cd2+ ion. The metallated rhMT structures were shown to have very stable conformations, but only when fully metallated. Two or more conformations were observed at low pH in the ESI-MS data, which are interpreted as arising from the presence of structure, as opposed to a random coil, in the apo-rhMT. This is the first report of the existence of a structure in the two-domain metal-free apo-MT proteins. Only at extremely low pH does the structure open fully to give the highest charge distribution, which is associated with a random coil. Pre-existing structural features in the apo-MT would explain why the metallation reactions occur so rapidly. [ABSTRACT FROM AUTHOR]

Abstract: Ganoderic acids are valuable bioactive secondary metabolites produced by a traditional medicinal mushroom Ganoderma lucidum (“Ling-zhi” in Chinese and “Reishi” in Japanese). In this work, a fast and efficient method for the recovery and purification of ganoderic acid T (GA-T) and ganoderic acid Me (GA-Me) from triterpene-enriched extracts of G. lucidum mycelia was developed by using reversed phase HPLC (RP-HPLC) on a C18 column with an acidified methanol–water mobile phase in combination with ultraviolet (UV) detection and electrospray ionization mass spectrometry (ESI-MS). The presence of each targeted GA (GA-T and GA-Me) in its corresponding peak was easily identified and confirmed by UV and MS. The chemical structures of the purified GA-T and GA-Me were further confirmed by 1H NMR. The retention behaviors of the two GAs over a temperature range of 15–55°C were also investigated. From the retention time data, van’t Hoff plots were obtained. The estimated enthalpy (ΔH) and entropy (ΔS) data suggest that the retention time difference between GA-T and GA-Me might be driven by an enthalpy difference. Furthermore, a semi-preparative HPLC purification was achieved on a semi-preparative C18 column using the conditions optimized for the analytical column. The method presented in this work can be a valuable tool for the rapid semi-preparative purification of targeted GAs, and it may also be applicable to some other natural products. [Copyright &y& Elsevier]

Abstract: Coupled size exclusion chromatography (SEC)–electrospray ionization mass spectrometry (ESI-MS) was applied to carefully map the product spectrum of a series of acrylate free radical polymerizations mediated via the reversible addition fragmentation chain transfer (RAFT) process. The product stream of a significantly rate retarded RAFT system (i.e. n-butyl acrylate (BA)/cumyl dithiobenzoate (CDB)) was compared with the less rate retarded RAFT polymerizations of BA mediated by cumyl phenyl dithioacetate (CPDA) and methyl acrylate (MA)/CPDA. In each case excellent agreement between the theoretical and experimental masses, as well as the simulated isotopic peak distributions, of polymeric species in the product stream was observed. Although conventional disproportionation and combination bimolecular termination products were clearly identified within the product spectra, the presence of irreversibly terminated RAFT intermediates, i.e. 3-armed star polymers, was not observed. The mass spectroscopic results are compared to modeling estimations (carried out via the PREDICI® program package) of the concentration ratios of 3-armed stars vs. conventional termination products. It is demonstrated that the occurrence of conventional termination products should be accompanied by a significant product stream associated with 3-armed star polymer material if cross termination was operational—at least under the current reaction conditions. The absence of three armed star polymer products in the polymers stream suggests that irreversible cross termination reactions may be of minor importance in the present systems. [Copyright &y& Elsevier]