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Human 5-lipoxygenase (5-LO) can form dimers as shown here via native gel electrophoresis, gel filtration chromatography and LILBID (laser induced liquid bead ion desorption) mass spectrometry. After glutathionylation of 5-LO by diamide/glutathione treatment, dimeric 5-LO was no longer detectable and 5-LO almost exclusively exists in the monomeric form which showed full catalytic activity. Incubation of 5-LO with diamide alone led to a disulfide-bridged dimer and to oligomer formation which displays a strongly reduced catalytic activity. The bioinformatic analysis of the 5-LO surface for putative protein-protein interaction domains and molecular modeling of the dimer interface suggests a head to tail orientation of the dimer which also explains the localization of previously reported ATP binding sites. This interface domain was confirmed by the observation that 5-LO dimer formation and inhibition of activity by diamide was largely prevented when four cysteines (C159S, C300S, C416S, C418S) in this domain were mutated to serines

During the last decade of the 20th century, the field of mass spectrometry has seen a revolutionary change in its application and scope. The introduction of soft ionization methods for the analysis of biological molecules has expanded the area of mass spectrometry from its early roots in the analysis of inorganic and organic species into the fields of biology and medicine. Today, the use of the mass spectrometry is extended to a wide range of applications in biotechnology and pharmaceutical industry, in geological, environmental and clinical research. In biochemistry, the principles of mass spectrometry are, however, broadly applicable in accurate molecular weight determination, reaction monitoring, amino acid sequencing, oligonucleotide sequencing and protein structure. In order to carry out their biological activities, proteins interact most often to each other and form transient or stable complexes. In addition, some proteins specifically interact also with other proteins or with non-protein molecules, such as DNA, RNA or metabolites, these interactions being critical for their function. Hence, defining the composition of protein complexes, as well as understanding how protein complexes are assembled and regulated yield invaluable insights into protein function. Coupled with an isolation technique to purify a specific protein complex of interest, mass spectrometry can rapidly and reliably identify the components of complexes. In addition, quantitative MS techniques offer the possibility of studying dynamically regulated interactions...., Im Laufe der letzten 10 Jahre des 20. Jahrhunderts hat sich die Massenspektroskopie sowohl in ihrer Anwendung als auch in ihrem Einsatzgebiet stark verändert. Durch die Einführung von schonenderen Ionisierungsmethoden für die Untersuchung biologischer Proben wurde die Massenspektroskopie nicht nur wie anfangs für anorganischen und organischen Moleküle, sondern auch in den Bereichen der Biologie und Medizin anwendbar. Heutzutage ist die Massenspektroskopie in einem weiten Feld wie in der Biotechnologie, Pharmazeutischen Unternehmen und in geologischen, ökologischen und klinischer Forschung angesiedelt. In der Biochemie wird die Massenspektroskopie unter anderem für die Auslesung der molaren Masse, das Überwachen von Reaktionen, die Bestimmung von Aminosäurensequenzen, Oligonukleotidsequenzen oder Proteinstrukturen verwendet. Um ihrer biologische Aktivität gerecht zu werden, treten häufig Wechselwirkungen zwischen Proteinen auf, welche Übergangsstrukturen oder feste Strukturen formen. Zusätzlich wechselwirken einige Proteine mit anderen Proteinen oder nicht aus Proteinen bestehenden Molekülen, wie zum Beispiel DNA, RNA oder Metaboliten. Diese Interaktion maßgeblich für die Funktion der Proteine. Deshalb bringen die Definition des Aufbaus von Proteinstrukturen und das Verständnis für deren Entstehung und Anordnung einen unverzichtbaren Einblick in die Funktion von Proteinen. Gekoppelt mit einer Isoalationstechnik, bei der das gewünschte Protein durch Aufreinigung gezielt extrahiert wird, kann die Massespektronomie schnell zuverlässig die einzelnen Bestandteile des Proteins bestimmen. Zusätzlich bieten quantitative Massenspektroskopiemessungen die Möglichkeit, dynamisch regulierte Wechselwirkungen zu untersuchen...

Human 5-lipoxygenase (5-LO) can form dimers as shown here via native gel electrophoresis, gel filtration chromatography and LILBID (laser induced liquid bead ion desorption) mass spectrometry. After glutathionylation of 5-LO by diamide/glutathione treatment, dimeric 5-LO was no longer detectable and 5-LO almost exclusively exists in the monomeric form which showed full catalytic activity. Incubation of 5-LO with diamide alone led to a disulfide-bridged dimer and to oligomer formation which displays a strongly reduced catalytic activity. The bioinformatic analysis of the 5-LO surface for putative protein-protein interaction domains and molecular modeling of the dimer interface suggests a head to tail orientation of the dimer which also explains the localization of previously reported ATP binding sites. This interface domain was confirmed by the observation that 5-LO dimer formation and inhibition of activity by diamide was largely prevented when four cysteines (C159S, C300S, C416S, C418S) in this domain were mutated to serines