Novel Aspect: Global phosphoproteomic analysis of cytokine signaling in primary β-cells Introduction The insulin-producing β-cells of the pancreatic islets of Langerhans are targeted by aberrant immune system responses in diabetes mellitus involving cytokines, especially interleukin-1β (IL-1 β), which initiate apoptosis of the β-cells. As only limited amounts of primary β-cells can be isolated from model organisms like mouse and rat, global phosphoproteomic analysis of these signaling events by mass spectrometry has generally been unfeasible. We have therefore developed a strategy for characterization of cytokine-induced phosphorylation signaling in primary β-cells. By combining, down-scaling, and optimizing existing methods for phosphopeptide enrichment and fractionation, a very specific and sensitive new setup was established. Preliminary results have shown the potential of this strategy for comprehensive phosphoproteomic analysis in primary β-cells. Methods Islets of Langerhans from 5–7 day old Wistar Furth rats were isolated by handpicking after collagenase digestion of the pancreata. Islets were precultured for 7 days in complete RPMI medium + 10% fetal bovine serum. Islets were exposed to culture medium with or without IL-1 β for 10 min. After cellular lysis in 8M urea, the proteome was digested using Lys-C and trypsin, and stable isotope-labeled by reductive dimethylation for subsequent mass spectrometry-based quantification. Phosphopeptides were enriched by TiO2 chromatography and separated into monophosphorylated and multiphosphorylated peptide pools using Sequential elution from IMAC (SIMAC). The monophosphorylated sample was fractionated by microscale HILIC HPLC and all fractions analyzed by nanoLC-ESI-MS/MS on an LTQ-Orbitrap Velos using beam-type collision-induced dissociation in the HCD-cell. Preliminary Data We performed three biological replicates of control versus 10 min. IL-1β stimulation of 600, 500, and 800 randomly picked rat islets of Langerhans (approximately 110, 90, and 150 µg of protein per condition, respectively) using quantification by stable isotope labeling via reductive dimethylation. The highly effective enrichment and fractionation method combined with a sensitive LC-MS/MS setup resulted in the identification of 5,051, 4,299, and 5,856 unique phosphorylated peptides in each of the experiments (at 1% false-discovery rate), of which 2,808 were found in all three biological replicates (65% of the phosphopeptides identified from the least abundant sample). Preliminary analysis has shown fairly limited relative phosphorylation changes induced by the IL-1β stimulation. This could result from the lack of serum starvation of the islets, which is usually being performed in cell culture-based phosphoproteomic experiments to increase stimulus-induced phosphorylation changes by reducing the general cellular protein phosphorylation level. Therefore, further IL-1β stimulation experiments on serum-starved islets have been performed to validate the phosphorylation changes induced by the cytokine. Preliminary results indicate phosphorylation changes in response to the short-term IL-1β stimulation in for example the calcium/calmodulin-dependent protein kinase type II. This observation is in line with studies using inhibitors of this kinase, implicating it in IL-1β-mediated JNK signaling in β-cells. Furthermore, several cytoskeletal proteins exhibited phosphorylation increases, such as LIM and SH3 domain protein 1, microtubule-associated proteins 1B and 2, and A-kinase anchor protein 12. These changes could reflect IL-1β-induced regulation of cytoskeletal reorganization in β-cells by which this cytokine can modulate cell-matrix interactions during inflammation, a regulation shown in other cell types. Further data analysis is currently ongoing, and the collective results of the experiments will hopefully facilitate additional insights into the effect of IL-1β on pancreatic β-cells and thereby the underlying pathways responsible for β-cell apoptosis in diabetes mellitus.
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American Society For Mass Spectrometrys Annual Conference, 2012