The main objective of this work is to design and engineer nanopores that will be used in biopolymer analysis, with the ultimate goal of sequencing single proteins. The ability of sequencing proteins at the single-molecule level is important in industry and base science. Proteins are biomarkers linked to disease, hence devices capable of identify proteins are important in medical diagnostics. In addition, while all cells have the same DNA, the expression of proteins is unique. Hence, if we want to understand how a cell works, we need to understand how proteins are made and modified. To date there is no single-molecule technique to sequence proteins. Single-molecule sequencing is important because proteins exist in cells in highly variable and heterogeneous mixtures. And only single-molecule techniques can address this issues.
During the duration of the proposal, we have taken two approaches. One was to engineer nanopores for the de novo identification of proteins. In this respect, we have built a biological nanopores with an embedded peptidase. An unfoldase would then select, unfold and deliver proteins to a nanopore-peptidase. The latter would then identify the individual fragmented peptides. In the next step, we have shown that if the peptides are cleaved in a well-defined manner (e.g. after positively charged residues as for the digestion with trypsin), proteins can be identified. Finally, we showed that the sequential identification of peptides at the single-molecule would lead to the identification of 98% of proteins in the proteome.
The nanopore was also able to transport intact proteins across the nanopore. Hence, we also demonstrated that our system is capable of characterising single proteins during their intact transport across the nanopore. In this 'mode' the peptise activity is removed and the identification amino acid by amino acid might be possible.
In a second single-molecule identification approach, we discovered that engineered nanopores could identify very selectively hemoglobin in blood. Substitution of a single amino acid, a condition that occurs in hemophilic patients, could also be detected. This approach is amenable of real-time and single-molecule identification in complex biological samples.
