RT Journal Article
SR Electronic
T1 Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.01.04.475002
DO 10.1101/2022.01.04.475002
A1 Brian D. Reed
A1 Michael J. Meyer
A1 Valentin Abramzon
A1 Omer Ad
A1 Pat Adcock
A1 Faisal R. Ahmad
A1 Gün Alppay
A1 James A. Ball
A1 James Beach
A1 Dominique Belhachemi
A1 Anthony Bellofiore
A1 Michael Bellos
A1 Juan Felipe Beltrán
A1 Andrew Betts
A1 Mohammad Wadud Bhuiya
A1 Kristin Blacklock
A1 Robert Boer
A1 David Boisvert
A1 Norman D. Brault
A1 Aaron Buxbaum
A1 Steve Caprio
A1 Changhoon Choi
A1 Thomas D. Christian
A1 Robert Clancy
A1 Joseph Clark
A1 Thomas Connolly
A1 Kathren Fink Croce
A1 Richard Cullen
A1 Mel Davey
A1 Jack Davidson
A1 Mohamed M. Elshenawy
A1 Michael Ferrigno
A1 Daniel Frier
A1 Saketh Gudipati
A1 Stephanie Hamill
A1 Zhaoyu He
A1 Sharath Hosali
A1 Haidong Huang
A1 Le Huang
A1 Ali Kabiri
A1 Gennadiy Kriger
A1 Brittany Lathrop
A1 An Li
A1 Peter Lim
A1 Stephen Liu
A1 Feixiang Luo
A1 Caixia Lv
A1 Xiaoxiao Ma
A1 Evan McCormack
A1 Michele Millham
A1 Roger Nani
A1 Manjula Pandey
A1 John Parillo
A1 Gayatri Patel
A1 Douglas H. Pike
A1 Kyle Preston
A1 Adeline Pichard-Kostuch
A1 Kyle Rearick
A1 Todd Rearick
A1 Marco Ribezzi-Crivellari
A1 Gerard Schmid
A1 Jonathan Schultz
A1 Xinghua Shi
A1 Badri Singh
A1 Nikita Srivastava
A1 Shannon F. Stewman
A1 T.R. Thurston
A1 Philip Trioli
A1 Jennifer Tullman
A1 Xin Wang
A1 Yen-Chih Wang
A1 Eric A. G. Webster
A1 Zhizhuo Zhang
A1 Jorge Zuniga
A1 Smita S. Patel
A1 Andrew D. Griffiths
A1 Antoine M. van Oijen
A1 Michael McKenna
A1 Matthew D. Dyer
A1 Jonathan M. Rothberg
YR 2022
UL http://biorxiv.org/content/early/2022/01/05/2022.01.04.475002.abstract
AB 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.Competing Interest StatementAll authors affiliated with Quantum-Si, Inc, along with ADG and AMvO are shareholders of Quantum-Si, Inc.