Protein identification by nanopore peptide profiling

Nanopores are single-molecule sensors used in nucleic acid analysis, whereas their applicability towards full protein identification has yet to be demonstrated. Here, we show that an engineered Fragaceatoxin C nanopore is capable of identifying individual proteins by measuring peptide spectra that are produced from hydrolyzed proteins. Using model proteins, we show that the spectra resulting from nanopore experiments and mass spectrometry share similar profiles, hence allowing protein fingerprinting. The intensity of individual peaks provides information on the concentration of individual peptides, indicating that this approach is quantitative. Our work shows the potential of a low-cost, portable nanopore-based analyzer for protein identification.

Unidirectional Single-File Transport of Full-Length Proteins Through a Nanopore

Nanopore technology offers long, accurate sequencing of an DNA or RNA strand via enzymatic ratcheting of the strand through a nanopore in single nucleotide steps, producing stepwise modulations of the nanopore ion current. In contrast to nucleic acids, their daughter molecules, proteins, have neutral peptide backbones and side chains of varying charges. Further, proteins have stable secondary and higher order structures that obstruct protein linearization required for single file nanopore transport. Here, we describe a general approach for realizing unidirectional transport of proteins through a nanopore that neither requires the protein to be uniformly charged nor a pull from a biological enzyme. At high concentrations of guanidinium chloride, we find fulllength proteins to translocate unidirectionally through an a-hemolysin nanopore in a polymer-based membrane, provided that one of the protein ends is decorated with a short anionic peptide. Molecular dynamics simulations show that such surprisingly steady protein transport is driven by a giant electro-osmotic effect caused by binding of guanidinium cations to the inner surface of the nanopore. We show that ionic current signals produced by protein passage can be used to distinguish two biological proteins and the global orientation of the same protein (N-to-C vs. C-to-N terminus) during the nanopore transport. With the average transport rate of one amino acid per 10 μs, our method may enable direct enzyme-free protein fingerprinting or perhaps even sequencing when combined with a high-speed nanopore reader instrument.

Bacillus pumilus 15.1, a Strain Active against Ceratitis capitata, Contains a Novel Phage and a Phage-Related Particle with Bacteriocin Activity

A 98.1 Kb genomic region from B. pumilus 15.1, a strain isolated as an entomopathogen toward C. capitata, the Mediterranean fruit fly, has been characterised in search of potential virulence factors. The 98.1 Kb region shows a high number of phage-related protein-coding ORFs. Two regions with different phylogenetic origins, one with 28.7 Kb in size, highly conserved in Bacillus strains, and one with 60.2 Kb in size, scarcely found in Bacillus genomes are differentiated. The content of each region is thoroughly characterised using comparative studies. This study demonstrates that these two regions are responsible for the production, after mitomycin induction, of a phage-like particle that packages DNA from the host bacterium and a novel phage for B. pumilus, respectively. Both the phage-like particles and the novel phage are observed and characterised by TEM, and some of their structural proteins are identified by protein fingerprinting. In addition, it is found that the phage-like particle shows bacteriocin activity toward other B. pumilus strains. The effect of the phage-like particles and the phage in the toxicity of the strain toward C. capitata is also evaluated.

Chop-n-drop: in silico assessment of a novel single-molecule protein fingerprinting method employing fragmentation and nanopore detection

The identification of proteins at the single-molecule level would open exciting new venues in biological research and disease diagnostics. Previously we proposed a nanopore-based method for protein identification called chop-n-drop fingerprinting, in which the fragmentation pattern induced and measured by a proteasome-nanopore construct is used to identify single proteins. However whether such fragmentation patterns are sufficiently characteristic of proteins to identify them in complex samples remained unclear. In the simulation study presented here, we show that 97.9% of human proteome constituents are uniquely identified under close to ideal measuring circumstances, using a simple alignment-based classification method. We show that our method is robust against experimental error, as 78.8% can still be identified if the resolution is twice as low as currently attainable and 10% of proteasome restriction sites and protein fragments are randomly ignored. Based on these results and our experimental proof-of-concept, we argue that chop-n-drop fingerprinting has the potential to make cost-effective single-molecule protein identification feasible in the near future.