Geschmack ist vorhersagbar: Mit FlavorMiner. FlavorMiner heißt das Tool, das IPB-Chemiker und Partner aus Kolumbien jüngst entwickelt haben. Das Programm kann, basierend auf maschinellem Lernen (KI), anhand der…
Seit Februar 2021 bietet Wolfgang Brandt, ehemaliger Leiter der Arbeitsgruppe Computerchemie am IPB, sein Citizen Science-Projekt zur Pilzbestimmung an. Dafür hat er in regelmäßigen Abständen öffentliche Vorträge zur Vielfalt…
Palm-Forster, M. A. T.; Eschen-Lippold, L.; Lee, J.;A mutagenesis-based screen to rapidly identify phosphorylation sites in mitogen-activated protein kinase substratesAnal. Biochem.427127-129(2012)DOI: 10.1016/j.ab.2012.05.015
Identification and characterization of protein phosphorylation sites often requires mass spectrometric analysis, which is not trivial or accessible to many laboratories. Here, a targeted strategy to mutagenize putative phosphorylation sites within mitogen-activated protein kinase (MAPK) substrates is described. This employs a combination of standard type II with type IIs restriction enzymes to rapidly create individual or multiple phosphorylation site mutant versions of kinase substrates with high efficiency, thereby reducing the cost for screening mutated clones.
Publikation
Wirsing, L.; Naumann, K.; Vogt, T.;Arabidopsis methyltransferase fingerprints by affinity-based protein profilingAnal. Biochem.408220-225(2011)DOI: 10.1016/j.ab.2010.09.029
Precise annotation of time and spatial distribution of enzymes involved in plant secondary metabolism by gel electrophoresis are usually difficult due to their low abundance. Therefore, effective methods to enrich these enzymes are required to correlate available transcript and metabolite data with the actual presence of active enzymes in wild-type and mutant plants or to monitor variations of these enzymes under various types of biotic and abiotic stress conditions. S-Adenosyl-L-methionine-dependent O-methyltransferases play important roles in the modification of natural products such as phenylpropanoids or alkaloids. In plants they occur as small superfamilies with defined roles for each of its members in different organs and tissues. We explored the use of S-adenosyl-L-homocysteine as a selectivity function in affinity-based protein profiling supported by capture compound mass spectrometry. Due to their high affinity to this ligand it was possible to identify developmental changes of flower-specific patterns of plant natural product O-methyltransferases and corroborate the absence of individual O-methyltransferases in the corresponding Arabidopsis knockout lines. Developmental changes in the OMT pattern were correlated with transcript data obtained by qPCR.
Publikation
Schilling, S.; Hoffmann, T.; Wermann, M.; Heiser, U.; Wasternack, C.; Demuth, H.-U.;Continuous Spectrometric Assays for Glutaminyl Cyclase ActivityAnal. Biochem.30349-56(2002)DOI: 10.1006/abio.2001.5560
The enzymatic conversion of one chromogenic substrate, -glutamine-p-nitroanilide, and two fluorogenic substrates, -glutaminyl-2-naphthylamide and -glutaminyl-4-methylcoumarinylamide, into their respective pyroglutamic acid derivatives by glutaminyl cyclase (QC) was estimated by introducing a new coupled assay using pyroglutamyl aminopeptidase as the auxiliary enzyme. For the purified papaya QC, the kinetic parameters were found to be in the range of those previously reported for other glutaminyl peptides, such as Gln-Gln, Gln-Ala, or Gln-tert-butyl ester. The assay can be performed in the presence of ammonia up to a concentration of 50 mM. Increasing ionic strength, e.g., potassium chloride up to 300 mM, resulted in an increase in enzymatic activity of about 20%. This is the first report of a fast, continuous, and reliable determination of QC activity, even in the presence of ammonium ions, during the course of protein purification and enzymatic analysis.
Publikation
Ziegler, J.; Vogt, T.; Miersch, O.; Strack, D.;Concentration of Dilute Protein Solutions Prior to Sodium Dodecyl Sulfate–Polyacrylamide Gel ElectrophoresisAnal. Biochem.250257-260(1997)DOI: 10.1006/abio.1997.2248