Analysis of proteins related toautoimmune and inflammatorydiseases with focus on newenrichment methodologies and MALDI-TOF MS

Abstract: Proteins have been widely studied in biological sample analysis due to the association with diseases. In the present project, method and technique development was carried out on the analysis of two proteins, immunoglobulin G (IgG) and osteopontin (OPN). The changes in IgG glycosylation, and elevated plasma OPN levels have been reported to be associated with chronic inflammation and autoimmune diseases.Lab synthesized zeolitic imidazolate framework (ZIF) nanocomposites (paper I) and environmentally friendly wood materials (paper II) were successfully utilized for IgG glycopeptide enrichment in order to eliminate the interference caused by non‑glycopeptides. The quantification and identification of glycopeptides by matrix assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) was simplified using label-free internal standard, and intact glycopeptide identification without glycan release. The separation of enriched glycopeptides was further studied on capillary electrophoresis.Due to the low abundance of OPN in plasma, preconcentration of OPN is required prior to MS analysis. In paper III, a fast, cheap and antibody-free method was developed for recombinant human OPN (rhOPN) preconcentration from a complex mixture, human plasma, together with MALDI-TOF/TOF MS identification.In proteomics, enzymatic digestion of proteins is a very common step. In paper IV, the utilization of thiol-ene microchips (TE microchip) immobilized with trypsin provided fast digestion with residence time of only 10 s. Furthermore, the TE microchip linked with ascorbic acid could be used for IgG glycopeptide enrichment. These applications made TE microchips ready for multifunctional tasks in proteomics.Methods and techniques developed in the present project can be applied in the future for the study of biological samples, to investigate the possible relation between these proteins and autoimmune and inflammatory diseases, as well as other related diseases.