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

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.