Peptide-level analysis coupled with amino acid resolution is important to elucidate how proteins influence health and disease. Peptide-centric methodologies using liquid chromatography-tandem mass spectrometry (LC-MS/MS) are widely used to detect native and modified proteins. While LC-MS/MS stands as the dominant bottom-up proteomics technique, accessing LC-MS/MS and interpreting MS/MS spectra remains challenging. Due to the large capital expenditure and space required for LC-MS/MS, core facilities are often required for processing and analyzing protein samples. In addition, unexpected fragmentation patterns can preclude peptide detection, requiring additional interrogation of peptides for a more complete analysis. To address these hurdles, we demonstrate the ability of Quantum-Si’s Platinum™ next-generation benchtop protein sequencer to identify proteins in mixtures and discern endogenous modifications from instrument-related effects with single molecule resolution, complementing existing LC-MS/MS techniques. This dynamic protein sequencing approach employs a mixture of dye-labeled N-terminal amino acid recognizers (NAARs) and aminopeptidases to probe digested peptides. The order of recognizer binding and kinetic properties of recognition segments are analyzed to determine peptide sequence and associated proteins.
Here, we identify protein mixtures consisting of therapeutically relevant growth factors, cytokines, and secreted proteins. Moreover, kinetic signatures from NAARs not only reveal peptides that escape MS/MS mapping due to factors such as peptide length and pyroglutamate formation, but also enable differentiation between Asparagine (Asn) and deamidated peptide variants. These findings underscore Platinum as an alternative and complementary technique to LC-MS/MS for protein identification, peptide-level mapping, and monitoring critical quality attributes (CQAs) during product development.