High-resolution detection of post-translational modifications using single-molecule protein sequencing

Post-translational modifications (PTMs) play a critical role in regulating protein function and cellular processes. However, due to the diversity and complexity of the proteome, detecting and quantifying PTMs remains challenging. While conventional mass spectrometry and antibody-based methods provide valuable information, they are limited in their capacity to resolve and quantify PTMs at the amino acid level, particularly for PTMs with minimal differences in mass. The Quantum-Si Platinum ® and Platinum ® Pro sequencers are benchtop instruments designed for high-resolution, single-molecule protein sequencing. These instruments use engineered N-terminal amino acid recognizers to accurately determine the sequence composition of proteins, enabling the detection of PTMs and making advanced proteomic analysis accessible in a standard laboratory setting. In addition to leveraging the recognizer binding patterns, the recent development of PTM-specific binders further enhances the instrument's ability to detect and quantify PTMs by selectively targeting modified residues. This combined approach improves sensitivity and specificity, allowing researchers to quantify PTMs and obtain even more detailed PTM profiles on a single run. In this study, we investigated the application of single-molecule protein sequencing on Platinum Pro to detect and quantify multiple PTMs across diverse protein types. We demonstrate the detection of phosphorylation order at several sites within the anaplastic lymphoma kinase (ALK) protein, citrullination events on vimentin, asparagine deamidation as a marker of molecular ageing, and the identification and differentiation of aspartic acid stereoisomers in calmodulin sequences. This is the first study demonstrating the ability to resolve and quantify multiple PTMs on the Platinum Pro, enabling differentiation of chemical changes and stereochemical variants that, while subtle, can nevertheless have significant biological impacts. Platinum Pro can also be used to add proteoform characterization to biomarker studies, providing additional resolution not readily available in broad biomarker screening panels. We expect this technology to democratize advanced protein characterization, making the high-resolution detection of PTMs available to a broader scientific community.