טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentNir Iftach
SubjectCensing Single Ubiquitin Chains Using Solid State
Nanopores
DepartmentDepartment of Biomedical Engineering
Supervisor Professor Amit Meller
Full Thesis textFull thesis text - English Version


Abstract

Protein ubiquitination is a fundamental regulatory post-translational modification controlling intracellular signaling events, conserved in all eukaryotes. Ub is specifically conjugated to Lysine residues of the substrate, following a cascade of events. This system is vast and complex due to the ability of Ub to become attached to proteins as a monomer or as a chain of Ub moieties connected by various different linkages. The type of the Ub modification determines either the fate of the protein, modulates its localization or affects its function.

The ability to identify and characterize the length and type of Ub chains is important for basic biophysical research. To date, Ub chains have been characterized only by conventional biochemical assays, primarily separating the chains by their length using gel electrophoresis. Determination of Ub linkage specificity relies on the use of specific antibodies or by using Ub mutants. These methods, however, are labor intensive, and provide only limited information. Hence, there is a need to develop novel methods that provide accurate, rapid, and cost-effective proteomic characterization of Ub chains.

My project explored the feasibility of using solid-state nanopores to perform a purely electronic protein sensing method, allowing quick and highly informative single-molecule sensing of Ub chains. Translocations of biomolecules passing through a nanopore can be detected through analysis of transient changes to the ion current. The first aim of this project was to find the proper conditions to overcome the temporal resolution challenge posed by small protein translocations. Then we challenged the method by asking three main questions. First, we tried to differentiate Ub chains by their length. We were able to show that a monomer, a di-Ub and a penta-Ub chain have different translocation characteristics. Second, we pondered if two di-Ub chains, differing only by their chain linkage types, could be distinguished. Two main populations were detected with statistically significant differences, demonstrating that this novel technique has significant advantages over bulk measurements. The third question was whether the nanopore method could quantify the product accumulation following de-ubiquitination reaction of di-Ub chains. Counting this reaction using nanopores opens up the possibility of replacing the gel electrophoresis method by a single molecule method, which requires a few hundred copies of protein instead of at least 1013 copies. In summary, we were able to demonstrate the next generation discrimination of proteins. This project sets the foundation for a protein analysis method at the single-molecule level, using the nanopore platform.