Samenvatting
Nanopores have been successfully implemented for DNA sequencing. However, nanopore is much less employed for the protein analysis or sequencing due to the high order structure, different charge
conditions of protein, which is composed of 20 different amino acids. In this thesis, we proposed a new way to sequence protein with nanopore in a manner similar with traditional mass spectrometry sequencing. The
protein could be digested into peptides with enzymes and the peptide mass identified with nanopores instead of mass spectrometry. We named this system
a nanopore peptide mass identifier. Comparing with the traditional mass spectrometry detection, nanopore peptide identifier is a single molecule technique
which could improve the detection sensitivity, making it possible to detect the ultra-low abundance proteins and post translation modifications (PTMs). To make
the identifier feasible, we first worked on the method to capture all peptides with different charge conditions into the nanopores by using Fragaceatoxin C
(FraC) nanopores. Wild type FraC nanopores could detect peptides down to 10 AA but the peptide shorter than that translocated too fast to be
detected. Therefore, we also focused on engineering the size of FraC nanopores for small pores. More importantly, there should be a
correlation between the nanopore ionic current signal and peptides mass if we ought to identify an unknown peptide to make the nanopore peptide
mass identifier. Not limited to the protein sequencing endeavors, the two-component pleurotolysin nanopore (PlyAB) with a 5.5 nm constriction
site, has been reconstituted and engineered for large folded protein detection.
conditions of protein, which is composed of 20 different amino acids. In this thesis, we proposed a new way to sequence protein with nanopore in a manner similar with traditional mass spectrometry sequencing. The
protein could be digested into peptides with enzymes and the peptide mass identified with nanopores instead of mass spectrometry. We named this system
a nanopore peptide mass identifier. Comparing with the traditional mass spectrometry detection, nanopore peptide identifier is a single molecule technique
which could improve the detection sensitivity, making it possible to detect the ultra-low abundance proteins and post translation modifications (PTMs). To make
the identifier feasible, we first worked on the method to capture all peptides with different charge conditions into the nanopores by using Fragaceatoxin C
(FraC) nanopores. Wild type FraC nanopores could detect peptides down to 10 AA but the peptide shorter than that translocated too fast to be
detected. Therefore, we also focused on engineering the size of FraC nanopores for small pores. More importantly, there should be a
correlation between the nanopore ionic current signal and peptides mass if we ought to identify an unknown peptide to make the nanopore peptide
mass identifier. Not limited to the protein sequencing endeavors, the two-component pleurotolysin nanopore (PlyAB) with a 5.5 nm constriction
site, has been reconstituted and engineered for large folded protein detection.
| Originele taal-2 | English |
|---|---|
| Kwalificatie | Doctor of Philosophy |
| Toekennende instantie |
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| Begeleider(s)/adviseur |
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| Datum van toekenning | 28-nov.-2019 |
| Plaats van publicatie | [Groningen] |
| Uitgever | |
| Gedrukte ISBN's | 978-94-034-2184-1 |
| Elektronische ISBN's | 978-94-034-2183-4 |
| DOI's | |
| Status | Published - 2019 |