A new technique based on fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting can identify where specific amino acids and post-translational modifications are located in a single full-length protein molecule. FRET via DNA exchange (FRET X) enables discrimination of proteins with only subtle differences, owing to its ability to localize residues on intact proteins. Proteoform identification methods with high sensitivity, like FRET X, have the potential to open up new opportunities in proteomics research and diagnosis based on biomarkers.

The peer-reviewed research article, “Full-length single-molecule protein fingerprinting,” was published in Nature Nanotechnology.

Proteoform profiling with FRET X

Proteoform diversity is biologically significant, but current protein analysis methods are ineffective in distinguishing between different proteoforms. Accurate proteoform information can only be acquired when studying the protein of interest in its complete form, such as through affinity-based methods involving probes like antibodies. However, the abundance of available probes may result in these approaches having limited specificity. 

Although high-resolution native mass spectrometry (MS) and other bottom-up techniques have demonstrated their efficacy in analyzing proteoform profiles, they do not offer precise information regarding the sequence of co-occurring modifications. Alternative top-down fragmentation experiments necessitate significant sample volumes, high levels of purity, and extensive data analysis.

Researchers from the Kavli Institute of Nanoscience at Delft University of Technology developed a method for examining complete proteins at the level of individual molecules. Due to the retention of positional information, structures with identical mass, which may be indistinguishable in mass spectrometry, can be readily differentiated based on their distinct FRET efficiencies.

Subnanometer resolution can be achieved by using single-molecule FRET X to probe the amino acids individually, allowing for the fingerprinting of both intrinsically disordered proteins and folded globular proteins. The precise measurement of various forms in mixtures using a machine learning classifier and the location of two O-N-acetylglucosamine (O-GlcNAc) groups allowed for the accurate analysis of alpha-synuclein, a protein with an unknown structure. In addition, FRET X fingerprinting was demonstrated with globular proteins Bcl-2-like protein 1, procalcitonin, and S100A9.

Proteomic range

An obstacle in single-molecule proteomics is the cell’s fluctuating levels of distinct protein species. Given the wide range of variation in the proteome, which spans multiple magnitudes, more prevalent species can overshadow less common ones. Due to its high sensitivity to individual molecules and its capacity to identify multiple proteins in a single visual field, the FRET X technique can detect even the most scarce proteins.

Implementing future optimizations, such as automated acquisition and scanning stages, can enhance throughput and sensitivity further. Alternatively, the challenge presented by the wide range of protein levels can be addressed by implementing protein enrichment strategies to focus on specific targets. For the current investigation, fingerprinting required less than a femtomole of labeled protein; however, researchers suggest that by utilizing the available microfluidics technology, the sensitivity could increase by one or two additional orders of magnitude.

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