Abstract

The identification and quantification of proteins lags behind DNA-sequencing methods in scale, sensitivity, and dynamic range. Here, we show that sparse amino acid–sequence information can be obtained for individual protein molecules for thousands to millions of molecules in parallel. We demonstrate selective fluorescence labeling of cysteine and lysine residues in peptide samples, immobilization of labeled peptides on a glass surface, and imaging by total internal reflection microscopy to monitor decreases in each molecule's fluorescence after consecutive rounds of Edman degradation. The obtained sparse fluorescent sequence of each molecule was then assigned to its parent protein in a reference database. We tested the method on synthetic and naturally derived peptide molecules in zeptomole-scale quantities. We also fluorescently labeled phosphoserines and achieved single-molecule positional readout of the phosphorylated sites. We measured >93% efficiencies for dye labeling, survival, and cleavage; further improvements should enable studies of increasingly complex proteomic mixtures, with the high sensitivity and digital quantification offered by single-molecule sequencing.

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References

  1. 1.

    , & On the dependency of cellular protein levels on mRNA abundance. Cell 165, 535–550 (2016).

  2. 2.

    , , , & How low can you go? A current perspective on low-abundance proteomics. Trends Analyt. Chem. 93, 171–182 (2017).

  3. 3.

    et al. Performance evaluation of a hybrid linear ion trap/orbitrap mass spectrometer. Anal. Chem. 78, 2113–2120 (2006).

  4. 4.

    , , & Dynamic range of mass accuracy in LTQ Orbitrap hybrid mass spectrometer. J. Am. Soc. Mass Spectrom. 17, 977–982 (2006).

  5. 5.

    in Quantitative Proteomics (eds. Eyers, C.E. & Gaskell, S.) 3–21 (The Royal Society of Chemistry, Cambridge, 2014).

  6. 6.

    , & A theoretical justification for single molecule peptide sequencing. PLOS Comput. Biol. 11, e1004080 (2015).

  7. 7.

    , , , & Single-molecule protein sequencing through fingerprinting: computational assessment. Phys. Biol. 12, 055003 (2015).

  8. 8.

    et al. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling. Nat. Nanotechnol. 9, 466–473 (2014).

  9. 9.

    , , & Graphene nanopores for protein sequencing. Adv. Funct. Mater. 26, 4830–4838 (2016).

  10. 10.

    , , & Reading the primary structure of a protein with 0.07 nm3 resolution using a subnanometre-diameter pore. Nat. Nanotechnol. 11, 968–976 (2016).

  11. 11.

    Amino acid discrimination in a nanopore and the feasibility of sequencing peptides with a tandem cell and exopeptidase. RSC Advances 5, 30694–30700 (2015).

  12. 12.

    , , , & Single-molecule protein identification by sub-nanopore sensors. PLoS Comput. Biol. 13, e1005356 (2017).

  13. 13.

    Method for determination of the amino acid sequence in peptides. Acta Chem. Scand. 4, 283–293 (1950).

  14. 14.

    , , & Solution-phase and solid-phase sequential, selective modification of side chains in KDYWEC and KDYWE as models for usage in single-molecule protein sequencing. New J. Chem. 41, 462–469 (2017).

  15. 15.

    , , , & Application of sequenator analyses to the study of proteins. Biochemistry 11, 4493–4502 (1972).

  16. 16.

    & Phosphorylation and functions of the RNA polymerase II CTD. Genes Dev. 20, 2922–2936 (2006).

  17. 17.

    et al. Enhancement of phosphoprotein analysis using a fluorescent affinity tag and mass spectrometry. Rapid Commun. Mass Spectrom. 19, 2157–2162 (2005).

  18. 18.

    , & Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat. Rev. Genet. 14, 618–630 (2013).

  19. 19.

    et al. Detection of post-translational modifications in single peptides using electron tunnelling currents. Nat. Nanotechnol. 9, 835–840 (2014).

  20. 20.

    , & Unfoldase-mediated protein translocation through an α-hemolysin nanopore. Nat. Biotechnol. 31, 247–250 (2013).

  21. 21.

    , & Single-molecule site-specific detection of protein phosphorylation with a nanopore. Nat. Biotechnol. 32, 179–181 (2014).

  22. 22.

    , & Solid-phase sequencing of 32P-labeled phosphopeptides at picomole and subpicomole levels. Methods Enzymol. 201, 186–199 (1991).

  23. 23.

    , & Single-molecule sequencing of an individual human genome. Nat. Biotechnol. 27, 847–850 (2009).

  24. 24.

    & The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell. Proteomics 1, 845–867 (2002).

  25. 25.

    & Improved beta-elimination-based affinity purification strategy for enrichment of phosphopeptides. Anal. Chem. 75, 6826–6836 (2003).

  26. 26.

    Solid-phase Edman degradation: an automatic peptide sequencer. Eur. J. Biochem. 20, 89–102 (1971).

  27. 27.

    , & Efficient subpixel image registration algorithms. Opt. Lett. 33, 156–158 (2008).

  28. 28.

    , , , & A dual-mode single-molecule fluorescence assay for the detection of expanded CGG repeats in Fragile X syndrome. Mol. Biotechnol. 53, 19–28 (2013).

  29. 29.

    , , , & Membrane protein stoichiometry determined from the stepwise photobleaching of dye-labelled subunits. ChemBioChem 8, 994–999 (2007).

  30. 30.

    & A method for selecting the bin size of a time histogram. Neural Comput. 19, 1503–1527 (2007).

  31. 31.

    et al. Deconvolving single-molecule intensity distributions for quantitative microscopy measurements. Biophys. J. 92, 2926–2943 (2007).

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Acknowledgements

We thank B. Cannon and R. Russell for early assistance with single-molecule imaging, M. Gadush for assistance with peptide synthesis, I. Riddington, J. Dinser, and K. Suhr for assistance in mass spectrometry analysis of fluorescently labeled peptides, Z. Simpson and J. Rybarski for assistance with image analysis, A. Ellington for many fruitful discussions, and the Texas Advanced Computing Center for high-performance computing. This work was supported by fellowships from the HHMI (to J.S.) and NSF (DGE-1610403 to A.A.B.), and by grants from DARPA (N66001-14-2-4051 to E.V.A. and E.M.M.), NIH (DP1 GM106408, R01 GM076536, and R35 GM122480 to E.M.M.), CPRIT (to E.M.M.), and the Welch foundation (F-1515 to E.M.M. and F-0046 to E.V.A.).

Author information

Author notes

    • Joseph Marotta

    Present address: Luminex Corporation, Austin, Texas, USA.

    • Jagannath Swaminathan
    • , Alexander A Boulgakov
    • , Erik T Hernandez
    •  & Angela M Bardo

    These authors contributed equally to this work.

Affiliations

  1. Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.

    • Jagannath Swaminathan
    • , Alexander A Boulgakov
    • , Angela M Bardo
    • , Joseph Marotta
    •  & Edward M Marcotte
  2. Department of Chemistry, University of Texas at Austin, Austin, Texas, USA.

    • Erik T Hernandez
    • , James L Bachman
    • , Amber M Johnson
    •  & Eric V Anslyn
  3. Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA.

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Contributions

J.S., A.A.B., E.T.H., A.M.B., J.L.B., A.M.J., E.V.A., and E.M.M. designed and analyzed the experiments or interpreted the data. J.S., E.T.H., A.M.B., J.L.B., and J.M. performed the experiments. J.S., A.A.B., E.T.H., A.M.B., E.V.A., and E.M.M. wrote and edited the manuscript.

Competing interests

J.S., A.M.B., E.M.M., and E.V.A. are cofounders and shareholders of Erisyon Inc. J.S., E.M.M., and E.V.A. are co-inventors on granted US patent PCT/US2012/043769. J.S., A.A.B., E.T.H., J.L.B., A.M.J., E.V.A., and E.M.M. are co-inventors on pending US patent PCT/US2015/050099.

Corresponding authors

Correspondence to Eric V Anslyn or Edward M Marcotte.

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DOI

https://doi.org/10.1038/nbt.4278

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