Beyond the Genome: Advancing our Understanding of the Proteome with Next-Generation Protein Sequencing™

Protein information is a crucial component of multiomics workflows, offering insights that integrate genetic, transcriptomic, and proteomic data for a more comprehensive understanding of biological systems. Multiomics studies would greatly benefit from methods that enable direct protein detection and characterization with exceptional sensitivity. In this context, we present the unique capabilities of Next-Generation Protein Sequencing™ (NGPS™) on Quantum-Si’s Platinum® and Platinum® Pro instruments, which utilize 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, protein variants, and PTMs. In this study, we demonstrate the application of NGPS to identify unknown proteins, detect proteoform- specific variation, and enable multiplexed protein analysis via the use of protein barcodes. In light of these capabilities, we envision NGPS as a complementary and valuable addition to multiomics workflows.