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Frolov A.,Institute of Bioanalytical Chemistry | Frolov A.,Center for Biotechnology and Biomedicine | Bluher M.,University of Leipzig | Hoffmann R.,Institute of Bioanalytical Chemistry | Hoffmann R.,Center for Biotechnology and Biomedicine
Analytical and Bioanalytical Chemistry | Year: 2014

Glucose can modify proteins in human blood, forming early glycation products (e.g., Amadori compounds), which can slowly degrade to advanced glycation endproducts (AGEs). AGEs contribute significantly to complications of diabetes mellitus and, thus, represent markers of advanced disease stages. They are, however, currently unsuitable for early diagnosis and therapeutic monitoring. Here, we report sensitive strategies to identify and relatively quantify protein glycation sites in human plasma samples obtained from type 2 diabetes mellitus (T2DM) patients and age-matched nondiabetic individuals using a bottom-up approach. Specifically, Amadori peptides were enriched from tryptic digests by boronic acid affinity chromatography, separated by reversed-phase chromatography, and analyzed on-line by high-resolution mass spectrometry. Among the 52 Amadori peptides studied here were 20 peptides resembling 19 glycation sites in six human proteins detected at statistically significantly higher levels in T2DM than in the normoglycemic controls. Four positions appeared to be unique for T2DM within the detection limit. All 19 glycation sites represent promising new biomarker candidates for early diagnosis of T2DM and adequate therapeutic control, as they may indicate early metabolic changes preceding T2DM. [Figure not available: see fulltext.] © 2014 Springer-Verlag Berlin Heidelberg.

Stalmans S.,Ghent University | Wynendaele E.,Ghent University | Bracke N.,Ghent University | Knappe D.,Institute of Bioanalytical Chemistry | And 7 more authors.
Protein and Peptide Letters | Year: 2014

Infections by antibiotic-resistant bacteria are becoming a great risk for human health, leading to an urgent need for new efficient antibacterial therapies. The short, proline-rich antimicrobial peptides from insects gained a lot of interest as a potential antibacterial treatment, having a low toxicity profile and being mainly active against Gram-negative bacteria. To know whether these antimicrobial peptides can be used for the treatment of cerebral infections, the blood-brain barrier transport characteristics of these peptides were investigated. This study describes the results of the in vivo bloodbrain barrier experiments in mice, as well as the in vitro metabolic stability in mouse plasma and brain of apidaecin Api137, oncocin, drosocin and drosocin Pro5Hyp. The four investigated peptides showed a significant influx into the brain with a Kin ranging between 0.37 and 0.86 μL/g × min and brain distribution volumes of 19.6 to 25.8 μL/g. Only for drosocin, a significant efflux was determined, with a kout of 0.22 min -1. After entering the brain, oncocin was for approximately 80% trapped in the endothelial cells, while the other peptides reached the brain parenchyma for about 70%. All peptides were stable in plasma and brain during the experiments, with estimated metabolic half-lives ranging between 47 min and 637 min. We conclude that the investigated short, proline-rich antimicrobial peptides show an influx into the brain, which make them a promising antibacterial treatment of cerebral infections. © 2014 Bentham Science Publishers.

Anavi S.,Hebrew University of Jerusalem | Ni Z.,Institute of Bioanalytical Chemistry | Ni Z.,University of Leipzig | Tirosh O.,Hebrew University of Jerusalem | And 2 more authors.
Redox Biology | Year: 2015

Accumulating evidence suggests that fatty livers are particularly more susceptible to several pathological conditions, including hepatic inflammation, cirrhosis and liver cancer. However the exact mechanism of such susceptibility is still largely obscure. The current study aimed to elucidate the effect of hepatocytes lipid accumulation on the nuclear electrophilic stress. Accumulation of intracellular lipids was significantly increased in HepG2 cells incubated with fatty acid (FA) complex (1mM, 2:1 oleic and palmitic acids). In FA-treated cells, lipid droplets were localized around the nucleus and seemed to induce mechanical force, leading to the disruption of the nucleus morphology. Level of reactive oxygen species (ROS) was significantly increased in FA-loaded cells and was further augmented by treatment with moderate stressor (CoCl2). Increased ROS resulted in formation of reactive carbonyls (aldehydes and ketones, derived from lipid peroxidation) with a strong perinuclear accumulation. Mass-spectroscopy analysis indicated that lipid accumulation per-se can results in modification of nuclear protein by reactive lipid peroxidation products (oxoLPP). 235 Modified proteins involved in transcription regulation, splicing, protein synthesis and degradation, DNA repair and lipid metabolism were identified uniquely in FA-treated cells. These findings suggest that steatosis can affect nuclear redox state, and induce modifications of nuclear proteins by reactive oxoLPP accumulated in the perinuclear space upon FA-treatment. © 2014 The Authors.

Milic I.,Institute of Bioanalytical Chemistry | Milic I.,University of Leipzig | Griesser E.,Institute of Bioanalytical Chemistry | Griesser E.,University of Leipzig | And 11 more authors.
Analytical and Bioanalytical Chemistry | Year: 2015

The levels of nitro fatty acids (NO2-FA), such as nitroarachidonic, nitrolinoleic, nitrooleic, and dinitrooleic acids, are elevated under various inflammatory conditions, and this results in different anti-inflammatory effects. However, other multiply nitrated and nitro-oxidized FAs have not been studied so far. Owing to the low concentrations in vivo, NO2-FA analytics usually relies on targeted gas chromatography–tandem mass spectrometry (MS/MS) or liquid chromatography–MS/MS, and thus require standard compounds for method development. To overcome this limitation and increase the number and diversity of analytes, we performed in-depth mass spectrometry (MS) profiling of nitration products formed in vitro by incubating fatty acids with NO2BF4, and ONOO-. The modified fatty acids were used to develop a highly specific and sensitive multiple reaction monitoring LC–MS method for relative quantification of 42 different nitrated and oxidized species representing three different groups: singly nitrated, multiply nitrated, and nitro-oxidized fatty acids. The method was validated in in vitro nitration kinetic studies and in a cellular model of nitrosative stress. NO2-FA were quantified in lipid extracts from 3-morpholinosydnonimine-treated rat primary cardiomyocytes after 15, 30, and 70 min from stress onset. The relatively high levels of dinitrooleic, nitroarachidonic, hydroxynitrodocosapenataenoic, nitrodocosahexaenoic, hydroxynitrodocosahexaenoic, and dinitrodocosahexaenoic acids confirm the presence of multiply nitrated and nitro-oxidized fatty acids in biological systems for the first time. Thus, in vitro nitration was successfully used to establish a targeted LC–MS/MS method that was applied to complex biological samples for quantifying diverse NO2-FA. [Figure not available: see fulltext.] © 2015 Springer-Verlag Berlin Heidelberg

Vemula V.,Institute of Bioanalytical Chemistry | Vemula V.,University of Leipzig | Ni Z.,Institute of Bioanalytical Chemistry | Ni Z.,University of Leipzig | And 2 more authors.
Redox Biology | Year: 2015

Carbonylation is a generic term which refers to reactive carbonyl groups present in biomolecules due to oxidative reactions induced by reactive oxygen species. Carbonylated proteins, lipids and nucleic acids have been intensively studied and often associated with onset or progression of oxidative stress related disorders. In order to reveal underlying carbonylation pathways and biological relevance, it is crucial to study their intracellular formation and spatial distribution. Carbonylated species are usually identified and quantified in cell lysates and body fluids after derivatization using specific chemical probes. However, spatial cellular and tissue distribution have been less often investigated. Here, we report coumarin-hydrazide, a fluorescent chemical probe for time- and cost-efficient labeling of cellular carbonyls followed by fluorescence microscopy to evaluate their intracellular formation both in time and space. The specificity of coumarin-hydrazide was confirmed in time- and dose-dependent experiments using human primary fibroblasts stressed with paraquat and compared with conventional DNPH-based immunocytochemistry. Both techniques stained carbonylated species accumulated in cytoplasm with strong perinuclear clustering. Using a complimentary array of analytical methods specificity of coumarin-hydrazide probe towards both protein- and lipid-bound carbonyls has been shown. Additionally, co-distribution of carbonylated species and oxidized phospholipids was demonstrated. © 2015 The Authors.

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