Studies of the proteome would greatly benefit from methods that enable direct protein detection and peptide characterization with exceptional sensitivity. In this context, we present the unique capabilities of Next-Generation Protein Sequencing™ on Quantum-Si"s Platinum® instrument, which utilizes single-molecule protein sequencing to achieve detection of unknown proteins and protein variants. Individual peptides are digested from proteins, immobilized on a semiconductor chip, and probed by dye-labeled N-terminal amino acid (NAA) recognizers, followed by subsequent aminopeptidase cleavage to expose each NAA in the peptide for recognition. Through the recording and analysis of fluorescent intensity, lifetime, and binding kinetics of each NAA binding event, we can successfully identify proteins and detect PTMs.

To demonstrate the power of protein sequencing, we identified proteins from serum without prior knowledge, identified pathological PTMs, and utilized protein barcodes to screen and characterize proteins. We isolated proteins from human serum via immunoprecipitation or SDS-PAGE and correctly identified them from the sequencing data with high confidence by mapping to an 8,000-protein reference panel. Additionally, we demonstrated the power of Platinum to detect PTMs based on kinetic changes by detecting citrullination and dimethylation of arginine—two PTMs that play key roles in disease states such as cardiovascular disease, autoimmune disease, and cancer. Furthermore, we identified both the wild-type and single-amino-acid variants of both hemoglobin alpha and beta from clinical samples of heterozygous patients. Finally, we showcased the detection of peptide biomarkers along with the utilization of barcoding techniques to streamline protein engineering, antibody engineering, and enzyme engineering.

These results demonstrate the transformative potential of single-molecule protein sequencing using Platinum for comprehensive proteome analysis. Our technology enables the detection and characterization of proteins and peptides in complex mixtures and biofluids, as well as the detection of critical PTMs implicated in human pathophysiology. As proteomics continues to advance, Quantum-Si's platform offers a promising approach to drive new insights into protein research and disease mechanisms.