ProtSeq: towards high-throughput, single-molecule protein sequencing via amino acid conversion into DNA barcodes

Proteins are the main structural and functional components of cells, and their dynamic regulation and post-translational modifications (PTMs) underlie cellular phenotypes. Next-generation DNA sequencing technologies have revolutionized our understanding of heredity and gene regulation, but the complex and dynamic states of cells are not fully captured by the genome and transcriptome. Sensitive measurements of the proteome are needed to fully understand biological processes and changes to the proteome that occur in disease states. Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect PTMs with single-molecule sensitivity and precision. However current methods for studying the proteome lag behind DNA sequencing in throughput, sensitivity, and accessibility due to the complexity and dynamic range of the proteome, the chemical properties of proteins, and the inability to amplify proteins. Here, we demonstrate single-molecule protein sequencing on a compact benchtop instrument using a dynamic sequencing by stepwise degradation approach in which single surface-immobilized peptide molecules are probed in real-time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. By measuring fluorescence intensity, lifetime, and binding kinetics of recognizers on an integrated semiconductor chip we are able to annotate amino acids and identify the peptide sequence. We describe the expansion of the number of recognizable amino acids and demonstrate the kinetic principles that allow individual recognizers to identify multiple amino acids in a highly information-rich manner that is sensitive to adjacent residues. Furthermore, we demonstrate that our method is compatible with both synthetic and natural peptides, and capable of detecting single amino acid changes and PTMs. We anticipate that with further development our protein sequencing method will offer a sensitive, scalable, and accessible platform for studies of the proteome.

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A single-molecule electrical approach for amino acid detection and chirality recognition

Science Advances ◽

10.1126/sciadv.abe4365 ◽

2021 ◽

Vol 7 (10) ◽

pp. eabe4365

Author(s):

Zihao Liu ◽

Xingxing Li ◽

Hiroshi Masai ◽

Xinyi Huang ◽

Susumu Tsuda ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Theoretical Calculations ◽

Protein Sequencing ◽

Current Fluctuations ◽

Point Contacts ◽

Conductance Data ◽

Single Molecule Junctions

One of the ultimate goals of analytic chemistry is to efficiently discriminate between amino acids. Here we demonstrate this ability using a single-molecule electrical methodology based on molecular nanocircuits formed from stable graphene-molecule-graphene single-molecule junctions. These molecular junctions are fabricated by covalently bonding a molecular machine featuring a permethylated-β-cyclodextrin between a pair of graphene point contacts. Using pH to vary the type and charge of the amino acids, we find distinct multimodal current fluctuations originating from the different host-guest interactions, consistent with theoretical calculations. These conductance data produce characteristic dwell times and shuttling rates for each amino acid, and allow accurate, statistical real-time, in situ measurements. Testing four amino acids and their enantiomers shows the ability to distinguish between them within a few microseconds, thus paving a facile and precise way to amino acid identification and even single-molecule protein sequencing.

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A theoretical analysis of single molecule protein sequencing via weak binding spectra

10.1101/352310 ◽

2018 ◽

Cited By ~ 1

Author(s):

Samuel Rodriques ◽

Adam Marblestone ◽

Ed Boyden

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Amino Acid Identity ◽

Peptide Sequencing ◽

Protein Sequencing ◽

Single Amino Acid ◽

Weak Binding ◽

Binding Spectra ◽

Accuracy Determination

AbstractWe propose and theoretically study an approach to massively parallel single molecule peptide sequencing, based on single molecule measurement of the kinetics of probe binding [1] to the N-termini of immobilized peptides. Unlike previous proposals, this method is robust to both weak and non-specific probe-target affinities, which we demonstrate by applying the method to a range of randomized affinity matrices consisting of relatively low-quality binders. This suggests a novel principle for proteomic measurement whereby highly non-optimized sets of low-affinity binders could be applicable for protein sequencing, thus shifting the burden of amino acid identification from biomolecular design to readout. Measurement of probe occupancy times, or of time-averaged fluorescence, should allow high-accuracy determination of N-terminal amino acid identity for realistic probe sets. The time-averaged fluorescence method scales well to extremely weak-binding probes. We argue that this method could lead to an approach with single amino acid resolution and the ability to distinguish many canonical and modified amino acids, even using highly non-optimized probe sets. This readout method should expand the design space for single molecule peptide sequencing by removing constraints on the properties of the fluorescent binding probes.Author summaryWe simplify the problem of single molecule protein sequencing by proposing and analyzing an approach that makes use of low-affinity, low-specificity binding reagents. This decouples the problem of protein sequencing from the problem of generating a high-quality library of binding reagents against each of the amino acids.

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Introducing EzAAI: a pipeline for high throughput calculations of prokaryotic average amino acid identity

The Journal of Microbiology ◽

10.1007/s12275-021-1154-0 ◽

2021 ◽

Vol 59 (5) ◽

pp. 476-480

Author(s):

Dongwook Kim ◽

Sein Park ◽

Jongsik Chun

Keyword(s):

Amino Acid ◽

High Throughput ◽

Amino Acid Identity ◽

Acid Identity ◽

Average Amino Acid Identity

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Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

Materials Chemistry Frontiers ◽

10.1039/d1qm00875g ◽

2021 ◽

Author(s):

Xiaojia Jiang ◽

Mingsong Zang ◽

Fei Li ◽

Chunxi Hou ◽

Quan Luo ◽

...

Keyword(s):

Real Time ◽

High Throughput ◽

Single Molecule ◽

Single Molecule Detection ◽

Sensitive Detection ◽

High Throughput Analysis ◽

Throughput Analysis ◽

The Real ◽

Highly Sensitive ◽

Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

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Encoding Multiple Virtual Signals in DNA Barcodes with Single-Molecule FRET

Nano Letters ◽

10.1021/acs.nanolett.0c04502 ◽

2021 ◽

Vol 21 (4) ◽

pp. 1694-1701 ◽

Cited By ~ 1

Author(s):

Sung Hyun Kim ◽

Hyunwoo Kim ◽

Hawoong Jeong ◽

Tae-Young Yoon

Keyword(s):

Single Molecule ◽

Dna Barcodes ◽

Single Molecule Fret

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High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias

International Journal of Molecular Sciences ◽

10.3390/ijms22115513 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5513

Author(s):

Sander Plessers ◽

Vincent Van Deuren ◽

Rob Lavigne ◽

Johan Robben

Keyword(s):

Amino Acid ◽

Phage Display ◽

High Throughput ◽

High Throughput Sequencing ◽

Target Selection ◽

Acid Modification ◽

Phage Display Technology ◽

Amplification Bias ◽

Large Deletions ◽

Depth Analysis

The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins—AlkB and FTO—biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library. Nanopore sequencing of the entire display vector showed additional enrichment of large deletions overlooked by region-specific sequencing, and further impacted the interpretation of the obtained amino acid motifs. We could attribute enrichment of these corrupted clones to amplification bias due to arduous FTO display slowing down host cell growth as well as phage production. This amplification bias appeared to be stronger than affinity-based target selection. Recommendations are provided for proper sequence analysis of phage display data, which can improve motive discovery in libraries of proteins that are difficult to display.

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Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

International Journal of Molecular Sciences ◽

10.3390/ijms22031018 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1018

Author(s):

Hiroaki Yokota

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Single Molecule ◽

Underlying Mechanism ◽

Amino Acid Sequences ◽

E Coli ◽

X Ray Crystallography ◽

Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

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