Protein Sequencing, One Molecule at a Time

Abstract

Despite tremendous gains over the past decade, methods for characterizing proteins have generally lagged behind those for nucleic acids, which are characterized by extremely high sensitivity, dynamic range, and throughput. However, the ability to directly characterize proteins at nucleic acid levels would address critical biological challenges such as more sensitive medical diagnostics, deeper protein quantification, large-scale measurement, and discovery of alternate protein isoforms and modifications and would open new paths to single-cell proteomics. In response to this need, there has been a push to radically improve protein sequencing technologies by taking inspiration from high-throughput nucleic acid sequencing, with a particular focus on developing practical methods for single-molecule protein sequencing (SMPS). SMPS technologies fall generally into three categories: sequencing by degradation (e.g., mass spectrometry or fluorosequencing), sequencing by transit (e.g., nanopores or quantum tunneling), and sequencing by affinity (as in DNA hybridization-based approaches). We describe these diverse approaches, which range from those that are already experimentally well-supported to the merely speculative, in this nascent field striving to reformulate proteomics.

Keywords: DNA nanotechnology; SMPS; fluorescence; fluorosequencing; nanopores; proteomics; single-molecule protein sequencing.

Figure 1:. Sequencing-by-degradation approaches.

(a) Schematic of fluorosequencing, which uses single molecule microscopy to monitor the reduction in fluorescence from immobilized, fluorescently labeled peptides following consecutive rounds of Edman degradation. Panel a adapted with permission from ref. . (b) Example data from ref. showing a peptide with labeled cysteines (blue stars) undergoing fluorosequencing. Top: Peptide sequence after each successive Edman cycle. Middle: Fluorescence emitted from one peptide molecule across Edman cycles. Bottom: Fluorescence quantified across 675 individual molecules with the characteristic stair-step decreases indicating positions of the fluorescently labeled cysteines. (c) A proposed strategy to combine Edman degradation with amino acid detection using NAABs (8,83,99). In one possible implementation, a palette of NAABs might be chosen that have weak but varying affinity for different amino acids, allowing for peptide sequencing based on binding kinetics (83). (d) Schematic representation of the nanopore mass spectrometer under development by Stein and colleagues (13). As single peptides or proteins are fed through the pore a fragmentation source releases each amino acid which will ultimately be identified by an ion detector. It has been speculated that a UV-laser fragmentation source could be used to dissociate amino acids.

Figure 2:. Sequencing-by-transit approaches.

(a) Schematic of a nanopore peptide sequencing experiment. An external voltage (V) is applied on the trans side of the pore, and a voltage ground is applied on the cis side, represented by the symbol on the top left side of the panel. (b) Depiction of a typical current blockade. I₀ symbolizes the open pore ionic current, and I_b symbolizes the blockaded current. (c) Mean relative residual current (I_b/I₀) and its standard deviation from the measurement of X_R7 (X being any amino acid) peptides. (d) Schematic of the ClpX single-molecule protein sequencing approach. Donor dye-labeled ClpXP is immobilized on a slide surface, and an ssrA-conjugated peptide labeled with an acceptor dye is threaded through the complex. (e) A fluorescence time trace that captures the different moments of translocation. (i) The donor signal from Cy3-labeled ClpXP excited at 532 nm (top green trace). (ii) Appearance of acceptor signal due to acceptor-directed excitation at 633 nm (middle red trace) indicates the binding of the acceptor-labeled peptide to ClpXP. (iii) The fluorescence resonance energy transfer (FRET) signal (bottom blue trace) reports on the presence of the peptide in ClpP. (iv) Loss of fluorescent and FRET signals indicates the release of the peptide. Panels a-c adapted with permission from Reference (72). Panels d and e adapted with permission from Reference (31).

Figure 3:. Sequencing-by-affinity approaches.

(a) Depiction of the DNA-PAINT method and its proposed application to protein fingerprinting (24)(90). Proteins are first covalently labeled with docking DNA strands that contain sequences specific for the amino acids being labeled. Imaging DNA strands conjugated with fluorophores hybridize with docking strands for specific amino acids to provide compositional information about a peptide or protein. In principle, by linearizing a protein and visualizing by super-resolution microscopy, relative positional information for the labeled amino acids can also be obtained. (b) Depiction of the FRET X method (29) and an application to protein fingerprinting (54). Proteins are labeled with docking DNA strands in addition to an acceptor dye on the protein terminus, the latter serving as a fixed reference point. Imaging strands containing a donor dye are then hybridized onto the docking strands. The resulting FRET signals are proportional to distance, providing a distance histogram characteristic of the protein’s sequence and fold. (c) Depiction of the proposed DNA nanoscope protein fingerprinting method (33, 88). DNA strands are amplified in such a way as to record distances between pairs of oligonucleotide-conjugated amino acids, with the resulting histogram of pairwise distances giving information about the order and position of the labeled amino acids.