Single-molecule Peptide Sequencing Through Reverse Translation Of Peptides Into DNA

Editor’s note: with recent speculation about the potential habitability of other worlds and understanding the processes whereby other biospheres arise and evolve, thought needs to be given to developing sensor technology to do in situ analysis of samples collected by robotic systems and human crews. With size and power and energy constraints in mind, having the most compact way to quickly analyze a sample or monitor alien life forms will be of great utility to future astrobiology expeditions. Systems such as the one described below are good step in this direction. Such reverse translation/sequencing platforms also allow for proteins to be traced back to their original DNA sequencing.

Despite advances in mass spectrometry and emerging single-molecule approaches, sequencing peptides at the single-molecule level remains a central challenge in proteomics. Here we present a ‘reverse translation’ strategy that enables single-molecule peptide sequencing with single-amino-acid resolution.

In this approach, peptides undergo a modified Edman degradation that iteratively releases N-terminal amino acids tagged with peptide-specific DNA barcodes. Antibody-mediated proximity extension assays identify these barcoded amino acids and generate PCR-amplifiable DNA reporters that record the identity, position and originating peptide of each amino acid.

The resulting DNA library is directly read by high-throughput sequencing, converting peptide sequences into digital DNA outputs. Using this approach, we demonstrate true single-molecule peptide sequencing, achieving full sequence coverage in millions of reads and accurate differentiation of both native and post-translationally modified peptides.

These results establish a framework that redefines protein sequencing as a DNA sequencing problem and lays the foundation for high-throughput, de novo single-molecule protein sequencing.

Astrobiology, Genomics,