Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Liesinger, L.
Probing surface exposure of protein backbone and side chains employing HX-MS and a quantitative proteomics approach.
[ Diplomarbeit/Master Thesis ] Uni Graz; 2014. pp.106.
- Autor*innen der Med Uni Graz:
- Liesinger Laura
- Betreuer*innen:
- Birner-Grünberger Ruth
- Altmetrics:
- Abstract:
- Probing structural alterations of proteins by using LC – MS technologies is an expanding field in proteomic research. Two different approaches were investigated to obtain structural information about three model proteins: Lactate dehydrogenase (LDH), an IgG2 antibody and the complex forming S-layer protein SbsC. In the first approach, hydrogen deuterium exchange (HDX) which involves substitution of the backbone hydrogen atoms with deuterium due to an excess of deuterium in the protein solution was used. After quenching of the reaction and protein digestion, peptides were separated and measured by (tandem) mass spectrometry. Data was analyzed with HDX workbench to determine the degree of deuteration. Limitations of the technique were overlapping peptides with little deuterium incorporation due to extensive back exchange of the deuterium to hydrogen during the LC-MS run despite optimization of running conditions with respect to pH, temperature and time. Moreover, the sequence coverage remained too low. Thus overall insufficient structural information of the proteins could be obtained by this method. An alternative approach was therefore devised based on quantitative isotopic labeling of protein N-termini and lysines by dimethylation or isotopic coded protein ligands (ICPL). The native protein was compared to the denatured protein by labeling equal protein amounts in both states with different isotopic labels prior to measuring the combined sample by LC-MS/MS. Ratios of heavy to light labeled lysines were determined as a measure of the relative solvent accessibility of the residue in the native protein (complex). Quantitative results differed between dimethylation and ICPL indicating a dependency on size of the label and labeling conditions (pH, buffer) for each protein. However, a high degree of reproducibility within each of the two surface labels enables general application of the technique for structural analysis of proteins.