MO430MASS-SPECTROMETRIC IDENTIFICATION OF POST-TRANSLATIONAL GUANIDINYLATED PROTEINS IN THE CONTEXT OF SYSTEMIC LUPUS ERYTHEMATOSUS

Background and Aims With continuous identification of post-translational modified isoforms of proteins, it is becoming increasingly clear that post-translational modifications limit or modify the biological functions of native proteins are majorly involved in development of various chronic disease. This is mostly due to technically advanced molecular identification and quantification methods, mainly based on mass spectrometry. Mass spectrometry has become one of the most powerful tools for the identification of lipids. Method In this study, we used sophisticated high-resolution mass-spectrometric methods to analyze the soluble ligand of receptor Notch-3, namely the Y-box protein (YB)-1, in serum from systemic lupus erythematosus (SLE) patients. In addition, kidneys of lupus-prone (MRL.lpr) mice were analyzed by mass-spectrometric imaging techniques to identify the underlying pathomechanisms. Serum YB-1 was isolated by chromatographic methods, afterwards digested by trypsin and analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The kidneys were fixed in paraffin, then kidney sections were deparaffinized, tryptic digested and analyzed by mass-spectrometric imaging techniques. Mass-spectrometry of extracellular YB-1 in SLE patient serum revealed post-translational guanidinylation of two lysine’s within the highly conserved cold shock domain (CSD) of the YB-1 protein (YB-1-2G). Patients with increased disease activity and those with active renal involvement (lupus nephritis, LN) had a higher degree of dual-guanidinylation within the CSD. Of note, at least one of these modifications was present in all analyzed LN patients, whereas single-guanidinylated YB-1 was present in only one and double modification in none of the control individuals. Mass-spectrometric imaging analyses specifically localized YB-1-2G and increases Notch-3 expression in kidney sections from MRL.lpr mice. Results The data from this study clearly demonstrate the high potential of high-resolution mass spectrometric methods as well as mass spectrometric imaging techniques to identify pathomechanisms of diseases like SLE/LN.

Mass Spectrometric Imaging of Plasma Membrane Lipid Alteration Correlated with Amperometrically Measured Activity-Dependent Plasticity in Exocytosis

The mechanism of synaptic plasticity and its link to memory formation are of interest, yet relatively obscure, especially the initial chemical change in the cell membrane following transmitter release. To understand the chemical mechanism of plasticity, we studied how repetitive stimuli regulate certain membrane lipid species to enhance exocytotic release using mass spectrometric imaging. We found that increasing high-curvature lipid species and decreasing low-curvature lipids in the cell membrane favor the formation of a longer-lasting exocytotic fusion pore, resulting in higher release fraction for individual exocytotic events. The lipid changes observed following repetitive stimuli are similar to those after exposure to the cognitive enhancing drug, methylphenidate, examined in a previous study, and offer an interesting point of view regarding the link between plasticity and memory and cognition.