Proteomics research relies heavily on mass spectrometry, which has emerged as the most prominent and widely utilized approach for the identification of proteins. However, progress is being made in other analytical methods for peptides and proteins. In this month PROTrEIN ITN’s Journal Club, we are taking a glance at the characterization of proteins with nanopores through a publication from the University of Groningen entitled “Protein identification by nanopore peptide profiling”¹.

Nanopores are naturally occurring protein channels in cell membranes through which molecules can pass. Nanopore sequencing and profiling approaches make use of these structures to identify molecules. The nanopore is embedded in a membrane that separates two chambers filled with an electrolyte solution. An electrical potential is then applied across the membrane, creating a current that flows through the nanopore. The analytes that travel through the pore disrupt the ion current, which can then be measured (Fig. 1). The principle behind nanopore sequencing is based on the fact that different molecules (nucleotides in the case of nanopore sequencing) have different sizes and shapes, and therefore induce distinct changes in the ion current. Currently, nanopore technologies are the most commonly used for the sequencing of DNA, but progress is being made in their utilization for the identification of peptides and proteins. 

In the presented article the authors use nanopores to characterize individual proteins following their digestion by trypsin. After digestion, peptides go into the nanopore analyser and generate a change in ion current when they pass through the pore. The protein information is collected in an excluded current spectrum, which is a histogram that summarizes the translocation events of all the peptides. Since each change in the ion current relates mainly to the analyte volumes, the resulting spectrum is a representation of the peptide volumes after protease digestion and can be used for the identification of the original protein.

The identifications obtained with the nanopore analyses are compared to the results collected with ESI-MS (electrospray ionization mass spectrometry). For the comparison, the peptides identified through the mass spectrometry analysis are mapped into an inferred excluded current spectrum. This spectrum is generated through a computational calibration algorithm and compared to the nanopore-obtained one through spectral matching techniques. 

Based on the performed comparisons, the authors observe that the reproducibility of the obtained spectra is quite high. Although for specific proteins the correlation between the nanopore-observed spectrum and the inferred one is lower, this could be due to the computational inference of the spectra based on the peptide mass rather than the volume, which is key in nanopore analyses.In conclusion, the authors observe that although the nanopore analyzers might require improvements in terms of resolution, they could constitute low-cost solutions for performing high-throughput analyses. They especially point out that this technique could offer considerable advantages when having small amounts of material, as is the case of low-abundant proteins or heterogeneous ones. For this reason, mass spectrometry and nanopore techniques could even be used in parallel, as they show different and complementary strength when it comes to protein identifications.

Q&A with Florian Lucas, the first author of the publication

Since nanopore technologies can identify single molecules and do not require large amounts of material, do you foresee a wider use of nanopore peptide profiling in single-cell proteomics?

Nanopore profiling gains its strength from the low sample volumes and high sensitivity able to pick up, previously undetected, unidentified contaminants in our filtered water supply². They may therefore find their way into some single-cell proteomic pipeline. However, their development is some years from mainstream application due to several engineering challenges remaining for the sensory technology. Nonetheless, these are inevitably solved in due time, as exemplified by commercial nanopore DNA sequencing devices.

More specifically, while single prokaryotic cells may provide currently unachievably small numbers of analytes, it is undeniable that nanopores can be used to detect proteolytic peptides from single eukaryotic cells. These can remain largely undiluted as the total measurement volumes of state-of-the-art nanopore chambers is less than 150 nanolitres, which is still amenable to down-scaling. However, the theoretical resolution of peptide profiling using nanopore will not be sufficient to extract single-protein information in such complex samples. Rather there are two future developments I foresee for the technology. Firstly, the nanopore can be coupled with upstream separation techniques such as liquid chromatography to reduce sample complexity. Secondly, motor proteins can (in theory) be attached on top of the nanopore to directly sequence full proteins (one-by-one), similar to nanopore DNA sequencing. This concept has been shown by the groups of G. Maglia and C. Dekker in two independent ways^{3, 4}.

The current resolution of nanopore technology does not seem to allow for the detection of ‘small’ modifications (e.g methylation). Do you think this approach can one day reach that level of accuracy?

This is an interesting question, as the answer cannot be expressed with a simple yes or no. It is important to acknowledge that nanopores do not detect the mass, rather, the displacement of ions. This makes them ideal for the detection of modifications that alter the ionic current and will therefore be able to detect some, but not all, modifications. Methylation in particular is a modification we can detect using nanopore DNA sequencing, and there is no doubt that this modification on peptides can be detected. Moreover, our recent work shows that glycans can be observed on peptides⁵. We have also previously shown that conformational differences between leucine and isoleucine can be discriminated⁶, and our colleagues displayed the discrimination of phosphorylated peptides⁷.

In the article, only one protein digest is analyzed at a time. How feasible would it be to scale the technique to more complex samples, and what would be the main challenges?

The field is still in the proof-of-concept phase, which is best compared to mass spectrometry in the 1980s. Even mass spectrometry as a stand-alone technique is unable to extract all information from highly complex samples. Instead, it builds on the synergy between analytical separation, e.g. liquid chromatography (LC) or capillary electrophoreses. These methods amplify the separation power to allow complex analysis. Unpublished results show that we can have a steady flow of several milliliters per minute across the nanopore. This is more than compatible with downstream flows of analytical LCs. However, we cannot exclude the possibility of nanopores sequencing proteins one by one when coupled with a motor protein, but this is still in its theoretical and very early proof-of-concept phase^{3, 4}.

References

1. Protein identification by nanopore peptide profiling. Florian Leonardus Rudolfus Lucas, Roderick Corstiaan Abraham Versloot, Liubov Yakovlieva, Marthe T. C. Walvoort and Giovanni Maglia. Nat Commun (2021) 12, 5795. DOI: 10.1038/s41467-021-26046-9

2. The Manipulation of the Internal Hydrophobicity of FraC Nanopores Augments Peptide Capture and Recognition. Florian Leonardus Rudolfus Lucas, Kumar Sarthak, Erica Mariska Lenting, David Coltan, Nieck Jordy van der Heide, Roderick Corstiaan Abraham Versloot, Aleksei Aksimentiev, and Giovanni Maglia. ACS Nano 2021 15 (6), 9600-9613. DOI: 10.1021/acsnano.0c09958

3. Bottom-up fabrication of a proteasome–nanopore that unravels and processes single proteins. Shengli Zhang, Gang Huang, Roderick Corstiaan Abraham Versloot, Bart Marlon Herwig Bruininks, Paulo Cesar Telles de Souza, Siewert-Jan Marrink, and Giovanni Maglia Nat. Chem. 2021 13, 1192–1199. DOI: 10.1038/s41557-021-00824-w

4. Multiple rereads of single proteins at single–amino acid resolution using nanopores. Henry Brinkerhoff, Albert S. W. Kang, Jingqian Liu, Aleksei Aksimentiev, and Cees Dekker. Science 2021 374, 1509-1513(2021). DOI:10.1126/science.abl4381

5. Quantification of Protein Glycosylation Using Nanopores. Roderick Corstiaan Abraham Versloot, Florian Leonardus Rudolfus Lucas, Liubov Yakovlieva, Matthijs Jonathan Tadema, Yurui Zhang, Thomas M. Wood, Nathaniel I. Martin, Siewert J. Marrink, Marthe T. C. Walvoort, and Giovanni Maglia. Nano Letters 2022 22 (13), 5357-5364. DOI: 10.1021/acs.nanolett.2c01338

6. In silico assessment of a novel single-molecule protein fingerprinting method employing fragmentation and nanopore detection. Carlos de Lannoy, Florian Leonardus Rudolfus Lucas, Giovanni Maglia, Dick de Ridder. iScience 2021 24 (10), 103202. DOI: 10.1016/j.isci.2021.103202.

7. Label-Free Detection of Post-translational Modifications with a Nanopore. Laura Restrepo-Pérez, Chun Heung Wong, Giovanni Maglia, Cees Dekker, and Chirlmin Joo. Nano Letters 2019 19 (11), 7957-7964. DOI: 10.1021/acs.nanolett.9b03134