Development of bioconjugatable chemical probes toward the fluoro-sequencing of protein
Embargo Date
2027-08-29
OA Version
Citation
Abstract
Next-generation sequencing technologies for DNA and RNA have transformed genomics and transcriptomics, respectively, enabling single-cell and even single-nucleus resolution. These breakthroughs have driven therapeutic innovation and provided profound insights into fundamental biology. However, proteomics at the single-cell or single-protein level has lagged behind, lacking comparable advancements in sequencing technologies and the ability to fully capture the entire proteome within its native context. This gap stems from the immense challenge of mapping the vast diversity of proteoforms, ranging from tens of thousands to millions per cell, using conventional affinity-based reagents like fluorescently labeled antibodies. These tools not only struggle to cover the proteome’s complexity but also fail to detect critical post-translational modifications (PTMs), which are essential for understanding protein function and regulation beyond genomic data. Peptide sequencing, on the other hand, offers an unbiased and comprehensive strategy for identifying proteoforms. Typically employing a combination of protein degradation and mass spectrometry, it provides a holistic view of the proteome. Yet, despite innovative approaches to preserve spatial context, achieving single-cell, single-molecule spatial proteomics using mass spectrometry remains a formidable challenge. Herein, I describe the development of a next-generation proteomics platform designed to sequence and fingerprint proteins at the single-peptide level. This is made possible by our novel bioconjugatable BODIPY-dye probe, a dye that selectively reacts with the N-termini of all available proteins and peptide fragments. Once covalently bound, this pan-amine reactive probe exhibits a distinct photophysical response depending on the specific amino acid residues at the ultimate and penultimate positions, including post-translational modifications. Following the fluorescent readout of the dye probe at the N-terminus, the dye can be photochemically removed, conduct novel N-degradation, and subsequently re-react the new N-terminus with the dye probe to achieve iterative fluoro-sequencing. Through iterative fluorescent reads unbiased identification of any protein could be obtained using fluoro-sequencing. What sets our OMNI-probe technology apart is its ability to identify all naturally occurring amino acids, including those with PTMs, ensuring comprehensive sequencing data and built-in error correction. In our presentation, we will highlight recent advancements in our OMNI-probe technology and its potential to create a fluoro-sequencing proteomics platform capable of fully digitizing the proteome.
Description
2025