Mass spectrometry is the current choice for sequencing proteins and for large-scale proteomic studies. However, mass spectrometry requires extremely sophisticated, bulky and expensive machines. Further, low abundance proteins and post-translational modifications are notoriously challenging to measure. Recently, nanopores have emerged as exciting new sensors for the low-cost and single-molecule sequencing of DNA. Herein, recent results showing that a nanopore can also be used to identify the mass of individual peptides in solution are presented. This approach will allow fabricating a portable peptide mass analyzer at extremely low cost.
The main technique to study and identify proteins is currently mass spectrometry. In a typical experiment in bottom-up proteomics, which is the large-scale study of proteins produced or modified by an organism or system, proteins are extracted and proteolytically digested into peptides and analyzed by MS, separated by liquid chromatography. This approach is very powerful and can be used to identify virtually all proteins. However, the throughput is generally low. Further, the identification of low abundance proteins, which can vary from a few to 109 copies per cell, and the characterization of their heterogeneity in post-translational modification remains a challenge. Finally, a mass spectrometer is expensive, complex, and cannot be easily miniaturized.
Biological nanopores are proteins that form tiny water conduits on a lipid membrane. Sharing the same principle of Coulter counters, under an externally applied potential the flux of ions across an individual nanopore generates an electrical signal that can be used to recognize single molecules entering the nanopore (Fig. 1). The output signal can be interfaced with low-cost and portable electronic devices. Most notably, nanopores are now used to sequence native DNA molecules in remote places such as the international space station.
Nanopores have also been used to detect polypeptides. The analysis of peptides with nanopores, however, have a new set of challenges compared to DNA. Polypeptides have 20 amino acids, non-uniform charge distributions, folded structures, heterogeneous sizes and shapes and might be heavily post-translationally modified.
The challenge, therefore, is to capture, unfold, translocate and identify peptides despite their chemical compositions.
Several nanopores have been used in peptide analysis. The authors of this article recently characterized Fragaceatoxin C (FraC) nanopores for biopolymer analysis . FraC resembles a truncated cone with a cis diameter of about 6.5 nm and a trans diameter of 1.6 nm (Fig. 1). The internal constriction of the nanopores is highly negatively charged, which makes the nanopore highly selective for cations. In turn, when a negative potential is applied to the trans side, a strong and directional cationic and water flow across the nanopore named electroosmotic flow (EOF) generates from cis to trans . Therefore, peptides inside the nanopore will be under the influence of an electrophoretic force, which depend on the charge of the peptide, and an electroosmotic force from cis to trans.
The size of a nanopore is important because it determines the range of analytes that can be detected. A challenge in nanopore analysis is that biological nanopores have a fixed size. Since only a small number of nanopores have been discovered thus far, one possibility of making nanopores with different sizes would be to control the nanopore assembly. FraC nanopore is made by the interaction of eight identical subunits. By modifying several key residues at the lipid binding interface, two smaller nanopores  (named type II FraC and Type III FraC, Fig. 2) have been prepared.
Uniform Capture and Translocation of Peptides
A nanopore-based peptide mass spectrometer should be able to capture peptides despite their chemical compositions. In nanopore analysis this is a challenge, because an electric field must be applied across the nanopore in order to generate the ionic signal output, and molecules enter the nanopore according to the direction of the applied field. This is advantageous to capture and stretch uniformly charged molecules such as DNA, but it is problematic when analyzing peptides, which have a wide distribution of charges. Using FraC nanopores, it was found that by reducing the negative charge of the peptides by lowering the pH of the solution to 4.5, the electroosmotic flow dominates the transport process and allows to capture peptides irrespectively of their chemical compositions .
Recognition of Peptides by Ionic Blockades with 44 Da Resolution
In nanopore sensing, several parameters in the electric signal can be used to evaluate a peptide including the ionic current during a blockade (IB), its duration, or the fluctuation of the signal (noise). Among these features, the IB is the most widely used. Initial experiments with the FraC nanopores revealed that peptides differing by one extra amino acid can be detected (e.g. angiotensin I, II, III and IV, Figure 1). The resolution limit of the nanopore sensor was further challenged by analysing identical peptides differing by the substitution of one amino acid. Angiotensin II and angiotensin A, which differ by 44 Da, appeared as distinctive signals .
At present, the nanopore system falls short from the resolution of commercial mass spectrometers. However, the technology is young and improvements are to be expected. For example, the limited resolution is mainly due to a relatively high spread of the blockade events, which is most likely due to thermal fluctuations, peptide conformations or peptide/nanopore interactions. Thus, optimization of these conditions and lowering the peptide translocation speed should improve the signal.
A Peptide Mass Spectrometer
The final aim of the work presented in this article was to develop a nanopore mass spectrometer for peptides. In order to achieve this goal, however, there must be a direct correlation between the nanopore signal and peptide mass. In nanopore sensing the ionic signal is generated by the number of ions that translocate through a nanopore per unit of time. When an analyte excludes a certain volume inside the nanopore, this excluded volume is reflected on the ionic current blockades. Therefore, the volume rather than the mass of an analyte is measured.
A series of peptides were tested using the three different types of FraC nanopores and was plotted the excluded current [(IO-IB)/IO] where IO is the current of the empty nanopore, as a function of the volume of the peptide. At pH 4.5 there is a direct correlation between the peptides’ mass and the signal only for positively charged peptides. The negatively charged peptides showed a lower excluded volume and faster residence times (Figure 3). After testing several conditions, it was found that at exactly pH to 3.8, all peptides showed a good correlation between the mass and the ionic signal . A likely explanation is that in order to be properly sampled, the peptide need to reach the constriction of FraC nanopore. At pH higher than 3.8, the electrostatic interactions between the negatively charged peptides and the negatively charged constriction prevent the peptide reaching the sampling region. At pH 3.8 the electrostatic interaction was greatly reduced allowing the sampling of the peptides within the same region (Fig. 3).
Since there is a correlation between the excluded current and the volume/mass of a peptide, FraC nanopores can be used to identify peptides without holding prior knowledge of their identity. The nanopores can hence be used as a peptide-volume identifier. Then the volume of the peptide can be converted to its mass. Although the resolution of the presented peptide mass analyzer is yet not comparable to commercial mass spectrometry devices, nanopores have distinctive advantages. Devices can be made containing arrays of thousands of single nanopores, which will allow a large detection output. Further, the analysis is single-molecule, hence heterogeneous protein solutions, post translational modifications and protein isoforms are more easily identified. Finally, a nanopore mass identifier recognizes peptides in biocompatible environments, allowing direct integration with other biological components. For example, if a protease-unfoldase pair is coupled directly above the nanopore sensor (Fig. 4), a nanopore system could be envisaged that identifies single protein in real-time.
Gang Huang1, Giovanni Maglia1
1Groningen Biomolecular Sciences & Biotechnology Institute, University of Groningen, Groningen, The Netherlands
Prof. Dr. Giovanni Maglia
Groningen Biomolecular Sciences & Biotechnology Institute, University of Groningen, Groningen,
Further articles on mass spectrometry!
1. Huang, G., Willems, K., Soskine, M., Wloka, C. & Maglia, G. Electro-osmotic capture and ionic discrimination of peptide and protein biomarkers with FraC nanopores. Nat. Commun. 8, 1–13 (2017).
2. Huang, G., Voet, A. & Maglia, G. FraC nanopores with adjustable diameter identify the mass of opposite-charge peptides with 44 dalton resolution. Nat. Commun. 1–10 (2019). doi:10.1038/s41467-019-08761-6