Researchers are exploring whether these ubiquitous fluorinated molecules might worsen infections or hamper vaccine effectiveness.
Image credit: Shutterstock/Dmitry Naumov.
Edited by Alan R. Fersht, Gonville and Caius College, Cambridge, United Kingdom, and approved February 21, 2018 (received for review May 1, 2017)
Significance
Protein sequencing remains a challenge for small samples. A sensitive sequencing technology will create the opportunity for single-cell proteomics and real-time screening for on-site medical diagnostics. To resolve protein identity, we previously developed a computational algorithm that analyzes the ordered sequence of only two types of amino acids within a protein species. Through modification of a biological nanomachine, here, we developed single-molecule fluorescence technology to linearize protein molecules and to read signals from labeled amino acids in an ordered manner. This proof of concept of single-molecule fingerprinting will open the door to single-molecule protein sequencing and pave the road toward the development of a new, fast, and reliable diagnostic tool.
Abstract
Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study challenging. Here, we demonstrate a proof of concept of single-molecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits unidirectional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.
Footnotes
- ↵1To whom correspondence may be addressed. Email: anne{at}annemeyerlab.org or c.joo{at}tudelft.nl.
Author contributions: J.v.G., A.S.M., and C.J. designed research; J.v.G., M.F., M.S., and P.T. performed research; J.v.G. and M.F. analyzed data; and J.v.G., M.S., A.S.M., and C.J. wrote the paper.
Conflict of interest statement: J.v.G., C.J., and A.S.M. hold a patent (“Single molecule protein sequencing”; WO2014014347).
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1707207115/-/DCSupplemental.
Published under the PNAS license.
Single-molecule peptide fingerprinting
Article Classifications
- Biological Sciences
- Applied Biological Sciences
Sign up for Article Alerts
Jump to section
You May Also be Interested in
Recent experiments and simulations are starting to answer some fundamental questions about how life came to be.
Image credit: Shutterstock/Radoslaw Lecyk.
Archaeologists have long tried to define the transition between the two time periods.
Image credit: Ceri Shipton.
Jonathon Yuly, David Beratan, and Peng Zhang investigate how electron bifurcation reactions work.
A study finds that giant pandas roll in horse manure to increase their cold tolerance.
Image credit: Fuwen Wei.