| Autor: |
Brian D. Reed, Michael J. Meyer, Valentin Abramzon, Omer Ad, Pat Adcock, Faisal R. Ahmad, Gün Alppay, James A. Ball, James Beach, Dominique Belhachemi, Anthony Bellofiore, Michael Bellos, Juan Felipe Beltrán, Andrew Betts, Mohammad Wadud Bhuiya, Kristin Blacklock, Robert Boer, David Boisvert, Norman D. Brault, Aaron Buxbaum, Steve Caprio, Changhoon Choi, Thomas D. Christian, Robert Clancy, Joseph Clark, Thomas Connolly, Kathren Fink Croce, Richard Cullen, Mel Davey, Jack Davidson, Mohamed M. Elshenawy, Michael Ferrigno, Daniel Frier, Saketh Gudipati, Stephanie Hamill, Zhaoyu He, Sharath Hosali, Haidong Huang, Le Huang, Ali Kabiri, Gennadiy Kriger, Brittany Lathrop, An Li, Peter Lim, Stephen Liu, Feixiang Luo, Caixia Lv, Xiaoxiao Ma, Evan McCormack, Michele Millham, Roger Nani, Manjula Pandey, John Parillo, Gayatri Patel, Douglas H. Pike, Kyle Preston, Adeline Pichard-Kostuch, Kyle Rearick, Todd Rearick, Marco Ribezzi-Crivellari, Gerard Schmid, Jonathan Schultz, Xinghua Shi, Badri Singh, Nikita Srivastava, Shannon F. Stewman, T.R. Thurston, Philip Trioli, Jennifer Tullman, Xin Wang, Yen-Chih Wang, Eric A. G. Webster, Zhizhuo Zhang, Jorge Zuniga, Smita S. Patel, Andrew D. Griffiths, Antoine M. van Oijen, Michael McKenna, Matthew D. Dyer, Jonathan M. Rothberg |
| Rok vydání: |
2022 |
| DOI: |
10.1101/2022.01.04.475002 |
| Popis: |
SummaryProteins 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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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