Methodology for the detection of the small, hydrophilic N-terminal peptide of S100A9 in the inflammatory diseases familial mediterranean fever and systemic-onset juvenile rheumatoid arthritisAlena Dreiling, Thomas Vogl, Johannes Roth, Simone Königpp 123-133Abstract:Elevated serum levels of calcium-binding S100A9 protein were found in patients with various inflammatory diseases such as familial mediterranean fever and systemic-onset juvenile rheumatoid arthritis. S100A9 purified from granulocytes of healthy individuals is present in several forms including an acetylated full-length form. Only this protein form contains an active cysteine while N-terminally truncated versions lack this cysteine and therefore cannot form disulfide bridges. A method was developed to detect the S100A9 N-terminal tryptic peptides (synthetic and native) using mass spectrometry in order to be able to reliably characterize the various protein forms in patient’s sera.Characterization of vitellogenin from the silkworm Bombyx moriNitin K. Singh, Britto C. Pakkianathan, Manish Kumar, Tulika Prasad, Simone König, Muthukalingan Krishnanpp 135-148Abstract:In silkworm, Bombyx mori, there are two types of fat body (FB) tissues, peripheral (PP) and perivisceral (PV)FB of which PPFB was commonly assumed to be the protein synthesis, and PVFB the storage site. The present study demonstrated that this hypothesis did not match experimental evidence for the lipid transfer protein vitellogenin (Vg). It showed that major biosynthesis of B. mori Vg takes place during the active feeding stage starting at day 3 V instar with PVFB as synthesis site. Maturation of Vg could also be observed in the increased appearance of its light and heavy chain at day 6 V instar. Vg was isolated from PVFB of day 3 of pupa. Vg subunits were purified and characterized as lipoglycophosphoproteins.Aldosterone detection using C18-separation and Q-TOF target analysisKristina Kusche-Vihrog, Simone Königpp 149-156Abstract:Aldosterone detection is of interest in clinical chemistry. Mass spectrometry-based methods are increasingly developed for ths purpose. Here, reversed-phase separation and gas phase fragmentation were used to investigate the sensitivity levels of aldosterone in biological buffers using a pseudo-multiple reaction monitoring (MRM) approach. Low fmol levels were reached by direct injection on column. Upstream ether extraction improved the detection limit 100fold.Thymidine analysis by pseudo-MRM LC-MS/MSAndre Kriegeskorte, Simone Königpp 157-159Abstract:Thymidine detection in biological buffer (culture media) based on reversed-phase separation and gas phase fragmentation was shown using a pseudo-multiple reaction monitoring (MRM) approach in positive ion mode. An acetonitrile/water/formic acid solvent system allowed the separation of the molecule early in the gradient. Low fmol sensitivity levels were reached.Insulin-regulated changes in cellular proteinnetworks and their phosphorylation statusUrsula Rescher, Ulli Martin Hohenester, Nina Quiskamp, Simone Königpp 161-258Abstract:Upon insulin stimulation of baby hamster kidney cells stably expressing the human insulin receptor (BHK-IR), tyrosine phosphorylation is high and persists for a long time. In order to screen for the global changes in this proteome upon 30 min stimulation, label-free quantification was performed. Fold changes of proteins were small. A shift in relative protein abundancies of, e.g., tubulins and proteins involved in protein biosynthesis and folding to higher concentrations was indicated in stimulated cells. Annexin A2 was also upregulated. In line with the insulin-induced drastic changes in F-actin organization in these cells, many proteins involved in the regulation of actin dynamics and/or linking membrane and the underlying actin cytoskeleton were found phosphorylated. Most interestingly, an isoform of the glucocorticoid receptor was detected phosphorylated upon insulin stimulation.

Nov. 12-13, 2013pp 259-272