Uncovering the Distinctive Topography of Individual Proteins and Proteoforms

Scientists have developed a new method for sequencing and identifying proteins at the single-molecule level, which could revolutionize the field of proteomics. The technique, called FRET X, uses a distance-based mapping strategy and single-molecule fluorescence resonance energy transfer (FRET) via DNA eXchange (FRET X) to create a full-length fingerprint of intact protein sequences.

FRET X works by attaching a DNA strand to the C-terminus of a protein, which can then transiently bind to a complementary sequence fused to the biomolecule of interest. A FRET donor and acceptor are then added, and the distance between them is measured using FRET. By optimizing the length of the DNA strands, the researchers were able to achieve a precision of ΔE ≈ 0.03 from ten docking events.

The researchers tested FRET X on a range of protein samples, including disordered alpha-synuclein (aSyn), globular proteins, protein biomarkers of bacterial and COVID-19 infections, and post-translationally modified aSyn. They were able to accurately identify the position of single cysteines along the aSyn protein, even when they were only five amino acids apart. They also demonstrated that FRET X could detect post-translational modifications (PTMs) such as O-GlcNAcylation.

One of the key advantages of FRET X is that it can analyze proteins from different sources, such as cell extracts and body fluids. The researchers propose a two-step bioconjugation reaction that enables the analysis of intact proteins with a solvent-exposed N-terminus group. They also demonstrate that pairing N- and C-terminal information increases the fingerprinting accuracy of aSyn to >85%.

The ability to fingerprint protein sequences at the single-molecule level is a significant leap forward for proteomics. While there is still work to be done to expand sequence coverage, integrate additional PTMs, and increase the distance range of observable residues to larger proteins, the development of FRET X represents a major breakthrough in the field.

The researchers believe that FRET X and other similar techniques will soon transform protein identification into a reality, just as they did for nucleic acids. The ability to analyze proteins at the single-molecule level will have numerous applications in fields such as medicine, biology, and biotechnology.

Source: <https://www.nature.com/articles/s41565-024-01638-w>