Identification and quantitative studies of protein immobilization sites by stable isotope labeling and mass spectrometry

A method was developed for characterizing immobilization sites on a protein based on stable isotope labeling and MALDI-TOF mass spectrometry. The model for this work was human serum albumin (HSA) immobilized onto silica by the Schiff base method. The immobilized HSA was digested by various proteolytic enzymes in the presence of normal water, while soluble HSA was digested in (18)O-enriched water for use as an internal standard. These two digests were mixed and analyzed, with the (18)O/(16)O ratio for each detected peptide then being measured. Several peptides in the tryptic, Lys-C, and Glu-C digests gave significantly higher (18)O/(16)O ratios than other peptides in the same digests, implying their involvement in immobilization. Analysis of these results led to identification of the N-terminus and several lysines as likely immobilization sites for HSA (e.g., K4, K41, K190, K225, K313, and K317). It was also possible from these results to quantitatively rank these sites in terms of the relative degree to which each might take part in immobilization. This method is not limited to HSA and silica but can be used with other proteins and supports.     

Global characterization of porcine intrauterine proteins during early pregnancy

Total protein secreted in the intrauterine lumen increases between day 10 and 13 post-estrus in both cyclic and pregnant gilts. The objective of this experiment was to identify those intrauterine proteins whose secretion changes during this time period. Sixteen mature gilts were either mated (day 0) or remained cyclic and were slaughtered at either day 10 or day 13 (n = 4 per status by day). At slaughter, each uterine horn was flushed with 20 ml Minimal Essential Medium. Flushings were dialyzed extensively against distilled water. A 0.5 ml aliquot of each was lyophilized, subjected to two-dimensional PAGE, and protein spots were identified following Coomassie staining of each gel. Densitometry was used to compare relative amounts of each spot. After statistical analysis, spots that differed due to either day, status, or day by status interaction were excised and digested in-gel with trypsin. The resulting peptides were analyzed by tandem mass spectrometry (MS/MS). Using MS/MS data, protein identification for each spot was attempted. There were 280 matching spots, of which 132 were significantly (P < 0.05 or 0.01) affected by pregnancy status, day, or the day by status interaction. Most (73%) spots increased from day 10 to day 13 with no effect of pregnancy. Several spots were identified as proteases or their inhibitors. Others potentially modify glycolipids and/or glycoproteins. These results indicate that the concentrations of many proteins within the intrauterine environment during early pregnancy are independent of the conceptus and could play roles in regulating the endometrial or conceptus glycocalyx.     

Quantification of character-impact odour compounds of roasted beef

Beef was roasted for 7 min in frying pans made of glass (A) or of stainless steel (B). The lower temperature in A yielded a more roasty-sweet flavour and the higher temperature in B a roasty-harsh, more caramellike flavour. Using stable isotope dilution assays the concentration levels of the odour compounds 4-hydroxy-2,5-dimethyl-3(2H)-furanone (I), 2-acetyl-2-thiazoline (II), 2-ethyl-3,5-dimethylpyrazine (III), 2,3-diethyl-5-methylpyrazine (IV), guaiacol (V) and methional (VI) were determined in samples A and B. The data obtained were divided by the corresponding odour thresholds which were evaluated nasally and retronasally. The results indicated that the flavour differences of A and B were preferentially caused by differences in the concentration levels of odorantsI, II, IV, andVI.

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Quantitative Analyse charakteristischer Geruchsstoffe von gebratenem Rindfleisch

Zusammenfassung Rindfleisch wurde 7 min in Pfannen aus Glas (A) und rostfreiem Stahl (B) gebraten. Die niedrigere Temperatur in A führte zu einem stärker röstig-süßen Aroma und die höhere Temperatur in B zu einem röstig-herben, stärker caramelartigen Aroma. Die Konzentrationen der Geruchsstoffe 4-Hydroxy-2,5-dimethyl-3(2H)-furanon (I), 2-Acetyl-2-thiazolin (II), 2-Ethyl-3,5-dimethyl-pyrazin (III), 2,3-Diethyl-5-methylpyrazin (IV), Guajacol (V) und Methional (VI) wurden in den Proben A und B mit Hilfe von Isotopenverdünnungsanalysen bestimmt. Die erhaltenen Werte wurden dividiert durch die entsprechenden nasal und retronasal ermittelten Geruchsschwellen. Die Ergebnisse zeigten, daß die Aromaunterschiede von A und B in erster Linie auf Unterschiede in den Konzentrationen der GeruchsstoffeI,II, IV undVI beruhen.

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Presented in part at the symposium Flavour Precursors, University of Würzburg, Sept. 30th to Oct. 2nd, 1992     

Structural analysis of peptide substrates for mucin-type O-glycosylation

The structures of three nine-residue peptide substrates that show differential kinetics of O-linked glycosylation catalyzed by distinct recombinant uridine diphosphate-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases) were investigated by NMR spectroscopy. A combined use of NMR data, molecular modeling techniques, and kinetic data may explain some structural features required for O-glycosylation of these substrates by two GalNAc transferases, GalNAc-T1 and GalNAc-T3. In the proposed model, the formation of an extended backbone structure at the threonine residue to be glycosylated is likely to enhance the O-glycosylation process. The segment of extended structure includes the reactive residue in a beta-like or an inverse gamma-turn conformation and flanking residues in a beta-strand conformation. The hydroxyl group of the threonine to be glycosylated is exposed to solvent, and both the amide proton and carbonyl oxygen of the peptide backbone are exposed to solvent. The exchange rate of the amide proton for the reactive threonine correlated well with substrate efficiency, leading us to hypothesize that this proton may serve as a donor for hydrogen bonding with the active site of the enzyme. The oxygens of the residue to be glycosylated and several flanking residues may also be involved in a set of hydrogen bonds with the GalNAc-T1 and -T3 transferases.     

Distributed phase-shift beamforming power balancing in ad-hoc and sensor networks

Ad-hoc and sensor networks are a well-studied area which gained a lot of attention in the research in the last decades. Two of the problems of battery-powered radio devices are limited transmitter power and finite amount of energy. This paper continues the path opened by the development of a new technology for radio communication which allows cluster communication beyond the horizon of each individual transmitter and the distribution of power need among the modules forming a cluster. This in terms decreases the average power need per device and contributes to a longer lifetime of the entire network.     

Sterol content in sunflower seeds (Helianthus annuus L.) as affected by genotypes and environmental conditions

Phytosterols play essential roles in many plant cell mechanisms. They are of industrial interest since, as part of the diet, they can reduce low density lipoprotein cholesterol. An increase in plant sterol contents, by improved crop varieties or crop management, could help to answer industrial demands and also to develop environmentally friendly extraction methods. The aim of this study was to evaluate genotypic variability of sterol content in cultivated sunflower and, in particular, effects of sowing date. Results showed large variability among a collection of sixteen sunflower inbred lines and hybrids. Total sterols varied almost twofold between extreme genotypes. A delay of sowing, giving higher temperatures during seed formation, induced a general increase in total sterol concentration by up to 35%, as well as variation in sterol composition according to genotype. These results are considered with an aim of improving sterol content by sunflower breeding programmes.