The Promise of Nanopore Technology: Advances in the Discrimination of Protein Sequences and Chemical Modifications

Abstract

ever, only 21 000 distinct protein-coding genes were identified. [2] Meaning just a tiny percentage of DNA has the information necessary for translation of all the proteins present in human cells. Among these genes, only 150 are systematic targets of somatic cancer mutations 2850 are drivers of rare diseases when mutated and ≈1100 are involved in diseases caused by multiple genes. [3] We understand now that mutations are not the only factor responsible for human disease. Epigenetics, another factor of interest responsible for disease, is influenced by several factors such as environment, aging, and lifestyle. One of the major sources of epigenetic changes is protein chemical modification. [4,5] Post-translational modifications consist of proteolytic cleavage or covalent addition of a chemical group to one or more amino acids, altering protein properties. [6] Eukaryote chemical modifications regulate many protein biological functions [7,8] such as apoptosis (cell death), epidermal growth factor receptor signaling, endocytosis, DNAdamage responses, and immunity. These modifications are also involved in a lot of human diseases: [9-11] cancer apparition and dissemination, autoimmune pathologies, neurodegenerative diseases or even type 2 diabetes. These chemical modifications are essential for therapeutic protein manufacturing. [12] More generally, the proteome has a key regulatory role in physiological processes that dictate disease development. [5] An immense number of distinct proteins, at least 10 6 different molecules, resulting from open reading frame (ORFs) variants, splice variants, and functional post-translational modifications generate the basis of the proteome's complexity. Moreover, the fluctuations in protein complexes throughout cell and life cycles and the subcellular localization of distinct proteins add further, often overlooked, complexity to the proteome. [5] We briefly describe the main factors explaining the proteome complexity in Figure 1. Because two-thirds of human genes are estimated to contain one or more alternative spliced exons and a large number of noncoding DNA sequences (introns), the alternative splicing mechanism leads to around 10 5 protein isoforms in eukaryotic cells. [13] This mechanism removes introns and associates combinations of exons together with multiple potential mature transcripts. Another factor increasing the complexity of a cell's protein pool is the variety of chemical modifications they are subject to and their rate. The main Only a small percent of human genomic DNA encodes for proteins. Additionally, protein isoforms variants and chemical modifications are not coded in the genome read by the cell machinery. The resulting protein diversity is deeply involved in regular and diseased cellular processes. One challenge for the field of biotechnology, after human genome sequencing, will be to decipher the proteome at a single molecule scale to analyze single-cell protein variability. In fact, cellular proteic information, often used as a source of biomarkers, is of grea...

Biological Nanopores: Engineering on Demand

“…Gaussian fits are used to determine the amplitudes of the blocked pore current and the most probable dwell time of the analyte, while the exponential fit is used to determine the mean interevent interval. For a detailed description of the processing of the collected data for nucleic acids and peptides, we refer the reader to references [114] and [115], respectively.…”

“…Furthermore, the time scale on which protein translocation occurs is too fast to resolve individual amino acids. Excellent and detailed reviews on challenges and advances in protein sequencing by nanopores can be found (see references [115,143,144]).…”

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“…[ 6,7 ] Nanopore technology has been a proven success for DNA sequencing and is making its first strides toward protein sequencing. [ 8–12 ] Aiming to discuss challenges, opportunities, and applications for the nanopore field and encourage cross‐disciplinary collaboration, [ 13–16 ] the 2020 Nanopore Weekly Meeting took place online every Monday starting from October 19, 2020 as a continuing international event after the 2020 Nanopore Electrochemistry Meeting, which was transformed to an online event in the past October due to the COVID‐19 pandemic. The 2020 Nanopore Weekly Meeting was chaired by Professor Mathias Winterhalter from Jacobs University (Germany), Professor Meni Wanunu from Northeastern University (USA) and Professor Yi‐Tao Long from Nanjing University (China) (Figure 1).…”

Real-time sensing of neurotransmitters by functionalized nanopores embedded in a single live cell

“…For nucleic acids, techniques have been established to determine the sequence or specific structures of both DNA and RNA [13][14][15][16][17]. Besides, single molecule sensing of proteins is another promising application of nanopore technology [18]. Various nanopore proteins have been used for detection of peptides [19,20], protein biomarkers [21][22][23][24][25], post-translational modifications [26], protein unfolding [27], and protein conformation [28].…”