The application of nanoelectrospray mass spectrometry on phosphoprotein analysis
As miniaturization of electrospray mass spectrometric technique continues to become the methodology of choice for the analysis of phosphorylation, significantly more types of conductive coatings for Nano-electrospray emitters are in demand. This dissertation investigated and compared the MS performance in the identification of phosphopeptides of three type's emitters -PANI, APTES treated PANI and hydrophobic acrylic treated PANI, developed in this laboratory. And over two order of magnitude greater LDR upper limit improvement than previous reported paper has been shown. In addition, the MS performance of protein mixture with two phosphopeptide enrichment mothods: IMAC (Ga III) and TiO 2 was studied in order to examine the coating materials effect factors potentially contributing to phosphopeptide identification. Moreover, In this study, we demonstrated that IL-17 signaling leads to phosphorylation of two sites in the C/EBPβ "regulatory 2 domain" in a sequential, interdependent fashion by using Nano-ESI mass spectrometry with homemade emitters. First rapid phosphorylation of Thr188 occurs within 15 minutes and is ERK-dependent. A second phosphorylation event targets Thr179, which requires prior Thr188 phosphorylation and GSK3β activity. We further show that the pathways activating C/EBPβ phosphorylation are mediated by distinct subdomains within IL-17RA. This is the first detailed dissection of the IL-17-mediated C/EBP pathway.