TY - JOUR AB - The aim of this study was to explore the impact of three different standard reference particulate matter (ERM-CZ100, SRM-1649, and SRM-2975) on epigenetic DNA modifications including cytosine methylation, cytosine hydroxymethylation, and adenine methylation. For the determination of low levels of adenine methylation, we developed and applied a novel DNA nucleobase chemical derivatization and combined it with liquid chromatography tandem mass spectrometry. The developed method was applied for the analysis of epigenetic modifications in monocytic THP-1 cells exposed to the three different reference particulate matter for 24h and 48h. The mass fraction of epigenetic active elements As, Cd, and Cr was analyzed by inductively coupled plasma mass spectrometry. The exposure to fine dust ERM-CZ100 and urban dust SRM-1649 decreased cytosine methylation after 24h exposure, whereas all three PM increased cytosine hydoxymethylation following 24h exposure, and the epigenetic effects induced by SRM-1649 and diesel SRM-2975 were persistent up to 48h exposure. The road tunnel dust ERM-CZ100 significantly increased adenine methylation following the shorter exposure time. Two-dimensional scatters analysis between different epigenetic DNA modifications were used to depict a significantly negative correlation between cytosine methylation and cytosine hydroxymethylation supporting their possible functional relationship. Metals and polycyclic aromatic hydrocarbons differently shapes epigenetic DNA modifications. AU - Cao, X. AU - Lintelmann, J. AU - Padoan, S. AU - Bauer, S. AU - Huber, A. AU - Mudan, A.* AU - Oeder, S. AU - Adam, T. AU - Di Bucchianico, S. AU - Zimmermann, R. C1 - 61314 C2 - 50148 CY - 525 B St, Ste 1900, San Diego, Ca 92101-4495 Usa TI - Adenine derivatization for LC-MS/MS epigenetic DNA modifications studies on monocytic THP-1 cells exposed to reference particulate matter. JO - Anal. Biochem. VL - 618 PB - Academic Press Inc Elsevier Science PY - 2021 SN - 0003-2697 ER - TY - JOUR AB - Extracellular vesicles (EVs) are cell-derived membrane-bound organelles that have generated interest as they reflect the physiological condition of their source. Mass spectrometric (MS) analyses of protein cargo of EVs may lead to the discovery of biomarkers for diseases. However, for a comprehensive MS-based proteomics analysis, an optimal lysis of the EVs is required. Six methods for the protein extraction from EVs secreted by the head and neck cell line BHY were compared. Commercial radioimmunoprecipitation assay (RIPA) buffer outperformed the other buffers investigated in this study (Tris-SDS, Tris-Triton, GuHCl, urea-thiourea, and commercial Cell-lysis buffer). Following lysis with RIPA buffer, 310 proteins and 1469 peptides were identified using LTQ OrbitrapXL mass spectrometer. Among these, 86% of proteins and 72% of peptides were identified in all three replicates. In the case of other buffers, Tris-Triton identified on average 277 proteins, Cell-lysis buffer 257 proteins, and Tris-SDS, GuHCl and urea-thiourea each 267 proteins. In total, 399 proteins including 74 of the top EV markers (Exocarta) were identified, the most of the latter (73) using RIPA. The proteins exclusively identified using RIPA represented all Gene Ontology cell compartments. This study suggests that RIPA is an optimal lysis buffer for EVs in combination with MS. AU - Subedi, P. AU - Schneider, M. AU - Philipp, J. AU - Azimzadeh, O. AU - Metzger, F. AU - Mörtl, S. AU - Atkinson, M.J. AU - Tapio, S. C1 - 56735 C2 - 47221 CY - 525 B St, Ste 1900, San Diego, Ca 92101-4495 Usa TI - Comparison of methods to isolate proteins from extracellular vesicles for mass spectrometry-based proteomic analyses. JO - Anal. Biochem. VL - 584 PB - Academic Press Inc Elsevier Science PY - 2019 SN - 0003-2697 ER - TY - JOUR AB - We report on the development of a novel assay protocol for the separation and detection of charge isoforms of DJ-1 in biological samples by automated capillary isoelectric focusing followed by immunological detection. DJ-1 (PARK7) is considered as a biomarker candidate for Parkinson's disease and may potentially support the differentiation of clinical subtypes of the disease. The new method allows for separation and subsequent relative quantitative comparison of different isoforms of DJ-1 in biological samples. The assay was successfully applied to the analysis of DJ-1 isoform patterns in brains from mice subjected to normal or high-fat diet and revealed statistically significant group differences. Furthermore, in a pooled and concentrated sample of human cerebrospinal fluid that was depleted of albumin and immunoglobulin G, four different charge variants of DJ-1 could be detected. Taken together, the capillary isoelectric focusing immunoassay for DJ-1 represents a promising tool that may ultimately serve in clinical biomarker studies. AU - Besong Agbo, D.* AU - Klafki, H.* AU - Poschmann, G.* AU - Genius, J.* AU - Janßen, C.* AU - Stühler, K.* AU - Wurst, W. AU - Meyer, H.E.* AU - Klingenspor, M.* AU - Wiltfang, J.* C1 - 28090 C2 - 32924 SP - 197-204 TI - Development of a capillary isoelectric focusing immunoassay to measure DJ-1 isoforms in biological samples. JO - Anal. Biochem. VL - 443 IS - 2 PB - Academic Press - Elsevier Science PY - 2013 SN - 0003-2697 ER - TY - JOUR AB - Mitochondrial dysfunctions decisively contribute to the progression of human diseases, implying that functional tests of isolated mitochondria may furnish conclusive information for diagnosis and therapy. Classical mitochondrial isolation methods, however, lack precisely adjustable settings for cell rupture, which is the most critical step in this procedure, and this complicates subsequent analyses. Here, we present an efficient method to isolate functionally active, intact mitochondria from cultured or primary cells and minute tissue samples in a rapid, highly reproducible manner. AU - Schmitt, S. AU - Saathoff, F.* AU - Meissner, L.* AU - Schropp, E.-M. AU - Lichtmannegger, J. AU - Schulz, S. AU - Eberhagen, C. AU - Borchard, S. AU - Aichler, M. AU - Adamski, J. AU - Plesnila, N.* AU - Rothenfusser, S.* AU - Kroemer, G.* AU - Zischka, H. C1 - 27180 C2 - 32567 SP - 66-74 TI - A semi-automated method for isolating functionally intact mitochondria from cultured cells and tissue biopsies. JO - Anal. Biochem. VL - 443 IS - 1 PB - Academic Press Elsevier PY - 2013 SN - 0003-2697 ER - TY - JOUR AB - Six alkyl ethers of 7-hydroxycoumarin, ranging from methoxy- to hexoxycoumarin, were studied for their NADPH-dependent metabolism by liver microsomes of male rats treated with phenobarbital (PB) or 3-methylcholanthrene (MC). The six alkyl ethers were metabolized by both types of microsomes, forming 7-hydroxycoumarin as the major product. Among the test compounds, 7-methoxycoumarin was unusual in that its dealkylation was inducible only by PB and not by MC. PB increased 7-methoxycoumarin-O-demethylase (MOCD) activity about four- to eightfold. Metyrapone strongly inhibited MOCD in PB-treated microsomes but not in MC-treated microsomes. Similarly, monoclonal antibodies directed toward PB-induced cytochrome P450s selectively suppressed MOCD in PB-treated microsomes. MOCD activity was observed in preparations of SD1 cells containing only cytochrome P450IIB1, while it was not found in preparations of XEM1 cells containing only cytochrome P450IA1. Demethylation of 7-methoxycoumarin was also mediated by the constitutive cytochrome P450 form(s) of liver, lung, small intestine, and kidney (in decreasing order). PB increased MOCD activity of small intestine by 40% but was without effect on the dealkylation activity of lung and kidney. MOCD activity was also detectable in differentiated rat hepatoma lines H4IIEC3 and 2sFou. The studies indicate that dealkylation of 7-methoxycoumarin is a highly sensitive, simple, and practical assay for estimating constitutive and PB-inducible cytochrome P450-dependent monooxygenase activities. AU - Reen, R.K.* AU - Ramakanth, S.* AU - Wiebel, F.J. AU - Jain, M.P.* AU - Singh, J.S.D.* C1 - 40812 C2 - 38538 SP - 243-249 TI - Dealkylation of 7-methoxycoumarin as assay for measuring constitutive and phenobarbital-inducible cytochrome P450s. JO - Anal. Biochem. VL - 194 IS - 2 PY - 1991 SN - 0003-2697 ER - TY - JOUR AB - A method for the isolation of RNA from different tissues of trees (seedlings, saplings, and adult trees) is described. Using this procedure it is possible to remove large amounts of disturbing polyphenolic compounds from nucleic acids. The method involves an acetone treatment of the freeze-dried and powdered plant material, the use of high salt concentrations in the extraction buffer and an aqueous two-phase system. These steps were combined with the conventional phenol/chloroform extraction and CsCl centrifugation. The method has been successfully applied to the isolation and purification of RNA from pine (Pinus sylvestris L. and Pinus mugo Turr.), Norway spruce (Picea abies L.), and beech (Fagus sylvatica L.). The functional quality of RNA extracted by this procedure has been characterized by its uv spectrum, by agarose gel electrophoresis with ethidium bromide staining, Northern blot hybridization, and in vitro translation. AU - Schneiderbauer, A. AU - Sandermann, H.J. AU - Ernst, D.E.W. C1 - 40850 C2 - 12236 SP - 91-95 TI - Isolation of functional RNA from plant tissues rich in phenolic compounds. JO - Anal. Biochem. VL - 197 IS - 1 PY - 1991 SN - 0003-2697 ER - TY - JOUR AB - A rapid and sensitive method for determining Cu-containing metallothionein (MT) is described. The main features of this Cd-saturation assay are: high-molecular-weight Cd-binding compounds are denatured with acetonitrile (50% final concentration), Cu bound to MT is removed with ammonium tetrathiomolybdate, excessive tetrathiomolybdate and its Cu complexes are removed with DEAE-Sephacel, apothionein is saturated with Cd, and excessive Cd is bound to Chelex 100. The thiomolybdate assay is capable of reliably detecting 14 ng MT and thus is particularly suitable for measuring MT in small tissue samples (e.g., biopsies), in extrahepatic tissues, and in cultured cells. Moreover, the combination of the thiomolybdate assay with the recently developed Cd-Chelex assay also makes it possible to determine the portion of MT which binds Cu (Cu load of MT), provided that the amount of non-Cu-thionein exceeds 100 ng, the detection limit of the Cd-Chelex assay. AU - Klein, D. AU - Bartsch, R. AU - Summer, K.H. C1 - 18965 C2 - 11356 SP - 35-39 TI - Quantitation of Cu-Containing Metallothionein by a Cd-Saturation Method. JO - Anal. Biochem. VL - 189 IS - 1 PY - 1990 SN - 0003-2697 ER - TY - JOUR AB - Oxidatively modified proteins have been implicated in a variety of physiologic and pathologic processes. Oxidative modification typically causes inactivation of enzymes and also the introduction of carbonyl groups into amino acid side chains of the protein. We describe a method to quantify oxidatively modified proteins through reduction of these carbonyl groups with tritiated borohydride. The technique was applied to purified, oxidatively modified glutamine synthetase and to bronchoalveolar lavage fluid from dogs and from humans. Since the protein content of lung lavage fluid is low, a very sensitive method was required to measure the oxidized residues. Reduction of the carbonyl group generated during oxidation of proteins with tritiated borohydride provided excellent sensitivity. Incorporation of tritium was directly proportional to the amount of protein with a range from 10 to 1000 micrograms. Should moieties other than amino acids be labeled, they are easily removed by rapid benchtop hydrolysis of the protein followed by chromatography on Dowex 50. AU - Lenz, A.-G. AU - Costabel, U. AU - Shaltiel, S. AU - Levine, R.L. C1 - 17766 C2 - 10674 SP - 419-425 TI - Determination of Carbonyl Groups in Oxidatively Modified Proteins by Reduction with Tritiated Sodium Borohydride. JO - Anal. Biochem. VL - 177 IS - 2 PY - 1989 SN - 0003-2697 ER - TY - JOUR AB - In order to determine free and conjugated pteridines, the pteridine-releasing procedure for biological materials (e.g., plasma, blood cells, bone marrow) was optimized and the parameters which influence pteridine release are discussed. Using reverse-phase high-performance liquid chromatography (rp-HPLC) on 5-μm ODS (C18; mobile phase, K2HPO4 buffer, pH 7.0-7.8), up to 10 pteridines (pterin-6-carboxylic acid, xanthopterin, neopterin, monapterin, isoxanthopterin, lumazine, biopterin, 6-hydroxymethylpterin, pterin) could be separated in the extracts of blood samples and determined fluorometrically (femtomolar range). In addition, three sepiapterins (sepiapterin, deoxysepiapterin, 3'-hydroxysepiapterin) could be separated with aqueous methanol as eluent (uv and fluorometric detection, respectively). The methods described are suited to compile pteridine patterns for plasma and individual cell fractions (erythrocytes, lymphocytes, buffy coat) from blood. Moreover, it was shown that the pteridine concentrations in dog blood (beagles) were in some cases substantially higher than in human blood, and that neopterin is lacking in the blood cell fractions of beagle dogs or occurs only in very low concentrations. AU - Andondonskaja-Renz, B. AU - Zeitler, H.J. C1 - 41032 C2 - 38294 SP - 68-78 TI - Separation of pteridines from blood cells and plasma by reverse-phase high-performance liquid chromatography. JO - Anal. Biochem. VL - 133 IS - 1 PY - 1983 SN - 0003-2697 ER - TY - JOUR AU - Zeitler, H.J. C1 - 42435 C2 - 35829 SP - 649-658 TI - N-methyl-2-anilino-6-naphthalenesulfonyl hydrazine (2,6-mansyl hydrazine) : A new sensitive fluorometric reagent for carbonyl compounds. JO - Anal. Biochem. VL - 88 IS - 2 PY - 1978 SN - 0003-2697 ER - TY - JOUR AB - Scenedesmus acutus contains about 10 major amines and at least 20 other amines which are present in very small quantities. The following amines were identified by mass spectrometry after separation of the trifluoroacetyl derivatives by gas-liquid chromatography and of the dansyl2 2 Abbrevlations used: dansyl, dimethylaminonaphthalinsulfonyl; TFA, trifluoroacetyl. derivatives by thin-layer chromatography: methylamine, dimethylamine, ethylamine, ethanolamine, putrescine, cadaverine, spermidine, N-(3-aminopropyl)-1,3-diaminopropane, N-(4-aminobutyl)-1,4-diaminobutane, 2-phenylethylamine, tyramine, piperazine, adenine, and γ-butyrolactam. The methods applied for the analyses of these amines are described and discussed. AU - Rolle, I. AU - Hobucher, H.E. AU - Kneifel, H. AU - Paschold, B. AU - Riepe, W. AU - Soeder, C.J. C1 - 42248 C2 - 35671 SP - 103-109 TI - Amines in unicellular green algae : 2. Amines in Scenedesmus acutus. JO - Anal. Biochem. VL - 77 IS - 1 PY - 1977 SN - 0003-2697 ER -