Single-Molecule Peptide Fingerprinting

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, such as single-cell analysis, challenging. Here we demonstrate a proof of concept of singlemolecule 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 uni-directional 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.\n\nSIGNIFICANCEProtein 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. In order 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 singlemolecule fingerprinting will open a new door to single-molecule protein sequencing and pave the road towards the development of a new, fast, and reliable diagnostic tool.