Master thesis in Wollscheid Lab

26.04.2016

Mass Spectrometric Analysis of Hepatitis B Virus X Proteoforms and Interactomes.

We are looking for a master student that has a basic education in molecular biology and proteomics and that would like to contribute to our efforts in analyzing different functional protein variants in the context of hepatitis B virulence using mass spectrometry.

Background

With 350 million chronic carriers and 620000 annual deaths (due to liver cirrhosis and hepatocellular carcinoma) hepatitis B virus (HBV) infections continue to be one of the major public health issues worldwide. The hepatitis B virus X-protein (HBx) is a 154 amino acid protein encoded by the HBX genome, which is known to have significant effects on HBV replication and pathogenesis. Through a direct interaction with host proteins, HBX is capable to promote HBV replication, regulate transcription of host genes and disrupt protein degradation.

Currently it is assumed that protein variants (so called proteoforms) produced by alternative splicing, proteolytic cleavage and post-translational modifications (PTMs) allow for the modulation of biological processes. In mass spectrometry, bottom-up proteomics is primarily used to identify and quantify peptides derived from proteins rather than the proteins themselves. Therefore, identified PTMs are restricted to individual peptides and we lack information about co-occurring modifications on the proteoform level. Top-down proteomics on the other hand is capable of identifying and quantifying single proteoforms (all combinations of PTMs and sequences on a single protein) through the analysis of intact proteins.

Goal

We want to analyze the modulation capabilities of the hepatitis B virus X-protein (HBX) in the context of HBV virulence. For that purpose we already generated different phosphorylation proteoforms of HBX and stably transfected them in HepRG2 cells.

Your job would be:

(i) to study the interactomes of the different known HBx proteoforms by affinity-purificatio mass spectrometry (AP-MS)

(ii) to develop and establish top-down strategies on a high-end mass spectrometer (Orbitrap Fusion) to identify new functional proteoforms of HBX. We will apply alternative fragmentation methods like electron capture/transfer dissociation (ECD/ETD) to enable an increased sequence coverage and PTM localization confidence on the protein level and employ state-of the art software tool (e.g. ProSight PC).

You will learn:

basic cell culture techniques, protein affinity purification, basic proteomics workflowsmass-spectrometry data handling and analysis. You will also have a chance to present your scientific results and contribute to a research publication.

If you are interested please contact Sandra Goetze () or Emanuela Milani ().

Literature

1. Gerlich, W. H. Medical virology of hepatitis B: how it began and where we are now. Virol. J. 10, 239 (2013).

2. Xie, N., Chen, X., Zhang, T., Liu, B. & Huang, C. Using proteomics to identify the HBx interactome in hepatitis B virus: how can this inform the clinic? Expert Rev. Proteomics 11, 59–74 (2014).

3. Bouchard, M. J. & Schneider, R. J. The enigmatic X gene of hepatitis B virus. J. Virol. 78, 12725–12734 (2004).

4. Smith, L. M., Kelleher, N. L. & Consortium for Top Down Proteomics. Proteoform: a single term describing protein complexity. Nat. Methods 10, 186–187 (2013).

5. Switzar, L., Giera, M. & Niessen, W. M. A. Protein digestion: an overview of the available techniques and recent developments. J. Proteome Res. 12, 1067–1077 (2013).

6. Brunner, A. M. et al. Benchmarking multiple fragmentation methods on an orbitrap fusion for top-down phospho-proteoform characterization. Anal. Chem. 87, 4152–4158 (2015).

7. Chait, B. T. Chemistry. Mass spectrometry: bottom-up or top-down? Science 314, 65–66 (2006).

 
 
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