Kombination von hochauflösender Biopolymer-Massenspektrometrie und Element-Massenspektrometrie in der Molekül- und Element-Proteomanalytik

Abstract

High-resolution biopolymer mass spectrometry was used for the characterisation of tau protein and its modifications, especially phosphorylation. The identification of phosphorylation sites of tau protein was performed with FTICR-MS in combination with the soft ionisation methods, MALDI and ESI. Thirty-three phosphorylation sites were found with MALDI-, chip-ESI-FTICR-MS and chip-ESI-FTICR-MS as well as with on-chip desalting. In the literature, only some of these phosphorylations were known.A new combination of biopolymer and elemental mass spectrometry using FTICR-MS and LA-ICP-MS (or ICP-MS) was developed to characterise modifications of proteins, e.g. phosphorylation and metal-binding in proteins. In addition to the measurement of phosphorylation and phosphorus concentration, metal concentrations in single proteins or protein mixtures separated by 2D gel electrophoresis could be determined. The modifications were analysed by FTICR-MS. Phosphorus and metal concentrations in protein mixtures were measured directly with good detection limits and high mass resolution in a short time by ICP-MS or LA-ICP-MS. This combination of two mass spectrometric methods was used to analyse mitochondrial proteins from baker's yeast and human brain proteins separated by 2D gel electrophoresis.Tracer experiments on Alzheimer brain proteins separated by 2D gel electrophoresis with enriched stable isotopes (65Cu, 67Zn und 54Fe) were used to analyse the stability of metal binding during gel electrophoresis. The Cu-containing and in part the Zn-containing proteins are stable during gel electrophoresis. An enrichment of 54Fe in Fe-containing proteins was observed.