The search of post-translational modified biomarkers for Diabetes type 1 in microparticles from Interleukin 1b stimulated beta cells

Description

Type 1 diabetes (T1DM) is characterized by selective destruction (apoptosis) of the insulin-producing beta-cells in the pancreatic islets of Langerhans, together with infiltration of the islets with lymphocytes and macrophages. The release of cytokines (e.g., interleukin 1b (IL-1b)) from those cells can stimulate the beta-cells to produce the free radicals like NO and reactive oxygen species which lead to cellular apoptosis. When cells undergo apoptosis or differentiation small vesicles termed microparticles (MPs) are released from the plasma membrane. These vesicles can eventually end up in the plasma or other biofluids and we suspect that they will contain biomarkers which are directly associated with the disease state.  It is presently not known if cell differentiation and apoptosis leads to similar MPs in terms of biomolecular composition. However, it is known that the mechanism behind MP formation in the two situations differs.

In this study we have developed an optimized protocol for the isolation of MPs from biofluids using dynamic laser scattering and flow cytometry and combined this with mass spectrometry (MS). We have identified and quantified the proteome from MPs originating from differentiating and IL-1b stimulated rat pancreatic beta-cells using stabile isotope labeling (SILAC). The highly enriched MPs where in addition treated directly with trypsin in order to shave off the external proteins. Sialic acid containing glycopeptides were recovered from this fraction using titanium dioxide (TiO₂) chromatography and identified and quantified using MS. In another experiment the protein content were submitted to trypsin digestion and the phosphorylated peptides were purified using TiO₂ and subsequently identified and quantified using MS.

Overall we have identified a large number of proteins and modified peptides from MPs and quantified those with respect to differentiation and apoptosis. We will combine the methods with peptide pre-purification and enhanced peptide fragmentation using ETD and multistage activation.