Periodic Reporting for period 4 - DeE-Nano (Design and Engineering Next-Generation Nanopore Devices for Bioplymer Analysis)
Periodo di rendicontazione: 2022-01-01 al 2022-06-30
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.
We also have shown that nanopore currents can be used to identify the volume of peptides. This is an unexpected and important discovery, as it open to the making devices that can compete with mass spectrometry devices. Using this discovery, we described a method for using nanopores to identify proteins.
Interestingly, we have e found that nanopores can identify enantiomer and diasterosomer differences in polypeptides. Finally, we have shown that nanopores can also be used to identify proteins and their modifications directly in complex biological samples.