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.

The production of fatty acids from simple nutrients occurs via a complex biosynthetic pathway with dozens of intermediate compounds and multiple branch points. Despite its importance for microbial physiology and biotechnology, critical aspects of fatty acid biosynthesis, especially dynamics of in vivo regulation, remain poorly characterized. We have developed a liquid chromatography/mass spectroscopy (LC-MS) method for relative quantification of fatty acid synthesis intermediates in Escherichia coli, a model organism for studies of fatty acid metabolism. The acyl carrier protein, a vehicle for the substrates and intermediates of fatty acid synthesis, is extracted from E. coli, proteolytically digested, resolved using reverse-phase LC, and detected using electrospray ionization coupled with a tandem MS. Our method reliably resolves 21 intermediates of fatty acid synthesis, with an average relative standard deviation in ratios of individual acyl-ACP species to total ACP concentrations of 20%. We demonstrate that fast sampling and quenching of cells is essential to accurately characterize intracellular concentrations of ACP species. We apply our method to examine the rapid response of fatty acid metabolism to the antibiotic cerulenin. We anticipate that our method will enable the characterization of in vivo regulation and kinetics of microbial fatty acid synthesis at unprecedented detail and will improve integration of fatty acid synthesis into models of microbial metabolism.