Abstract:
Mass spectrometric and chromatographic micro-methods have been developed and applied for the analysis of insulin-signalling phosphoproteins. The proteom-analytical methods (in-gel digest, protein identification) were first developed, investigated and applied to the analysis of the HIR-b-subunit. New RP- and IMAC micro tip techniques for the purification and enrichment of peptides and phosphopeptides were developed. Furthermore, the phosphopeptide specific neutral loss and precursor ion scan techniques (Triple-Quad-MS) have been investigated and compared. For the localization of phosphorylation sites NanoES-MS2 was investigated and a new MS3-based method (API- and q2-CID) was also developed. The scan techniques were applied for the identification of the phosphorylation sites from b-casein (< 5 pmol). For the efficient analysis of phosphopeptides off- and on-line-µIMAC/ES-MS-methods have been developed. Furthermore, a phosphopeptide specific alkaline µLC/ESI-API-CID hybrid scan method was developed which was applied for the analysis of b-casein. The LC- and MS-based methods were then used for the analysis of insulin-signalling phosphoproteins. Using [32P]-RP-HPLC phosphopeptide mapping we showed, that Ser1177/78/82 of the HIR were in vivo not essential for receptor autophosphorylation but necessary for substrate phosphorylation. The phosphorylation sites of the b-subunit of the in vivo insulin stimulated HIR-WT could be detected using m/z 79 precursor ion scanning. Using on-line UV-µLC/ESI-MS the in vitro substrate specifity of PKC isoforms was investigated in a semiquantitative way. In vitro with PKC-bI and -z treated GST-IRS-1Nk-fusionsprotein was analysed by using a new developed [32P]-2-D-RP-HPLC/micro-ESI/MS method and off-line NanoES-ion-trap-MS3. At Ser318 (IRS-1-sequence) a phosphorylation site was detected, which could play a major role in insulin signal transduction.