Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device

Abstract

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.

Identifying residues in unfolded whole proteins with a nanopore: a theoretical model based on linear inequalities

“…For two recent reviews of protein sequencing see [5,6]; a feature article [7] summarizes some ongoing developments in single-cell proteomics. The following are selected examples of work within the last decade or so in protein sequencing, including NIH-funded SMPS projects in progress: 1) Nanopores designed for SMPS [8]; 2) SMPS by sequential isolation and identification of N-terminal amino acids attached to a linker molecule [9]; 3) SMPS with nanopores and Raman Spectroscopy [10]; 4) Detecting single AA substitutions in individual molecules [11]; 5) unfoldase-coupled nanopore array technology [12] and protein arrays with epitopes for large-scale peptide recognition [13]; 6) recognition tunneling [14]; 7) theoretical methods such as transverse detection in a nanopore [15] and identification of cleaved residues with a tandem electrolytic cell (e-cell) [16]; and 6) sequencing of full-length proteins using N-terminal AA binders (NAABS) [17]. Almost all of the above are based on analog measurements.…”

Discovery of Amino Acid fingerprints transducing their amphoteric signatures by field-effect transistors

“…However, AAs are much smaller and can lay below the screening length. The new materials used in FET fabrication 14,15 and techniques of protein manipulation 5,[15][16][17] provide prospects for the analyses of low quantities of samples, providing a way to transduce the AA fingerprints. Our method is disruptive because in addition to providing resolution to distinguish different AAs is also able to read the signal from small polypeptides to discover mutations and provide information of the sequence.…”

Discovery of Amino Acid fingerprints transducing their amphoteric signatures by field-effect transistors

“…Using FETs to distinguish proteins by their isoelectric point and studying the variations of the surface potential (Ψo) was already considered by the inventor of the ISFET, P.Bergveld 17 but it was discarded because protein charges often lay above the Debye length and thus are screened by the ions in solution. AAs have a shorter lengt, new FET materials 18,19 and techniques of protein manipulation 7,9,19,20 provide prospects for the analyses of low quantities of samples, providing a way to transduce the AA fingerprints. Here, we used a combination of the Site-Binding with the Gouy-Chapman-Stern theories to calculate the signatures of AAs and small peptides expressed by Ψo and the total capacitance (CT) transduced with field-effect transistors (FETs).…”