Proteins exist as diverse proteoforms generated through post-translational modifications (PTMs), sequence variation, and structural heterogeneity. Conventional proteomic approaches typically rely on ensemble measurements that average signals across large populations of molecules, often obscuring molecular heterogeneity and rare protein states. Single-molecule fluorescence methods provide a means to directly observe individual molecular events and offer new opportunities for protein characterization. This thesis investigates fluorescence-based single-molecule approaches for the identification of protein PTMs, with a particular focus on phosphorylation and glycosylation. A pKa-guided cysteine labeling strategy was developed to enable selective modification of individual cysteine residues in proteins containing multiple reactive thiols, thereby improving site-specific labeling for fluorescence-based protein fingerprinting. To detect phosphorylation, a chemical conversion strategy based on β-elimination and Michael addition (BEMA) was applied to transform phosphoserine residues into chemically addressable sites suitable for fluorescent labeling, enabling site-specific detection of phosphorylation in peptides and α-synuclein at the single-molecule level. Single-molecule fluorescence methods were further applied to the analysis of protein glycosylation, demonstrating that interaction-based measurements can provide information on glycosylation-dependent molecular heterogeneity. The thesis further discusses current challenges in single-molecule proteomics, including sample preparation, labeling specificity, assay generality, and analysis in complex biological samples, and explores preliminary concepts for alternative detection strategies. Together, this work establishes experimental approaches that combine chemical modification and single-molecule fluorescence detection for the identification and characterization of protein PTMs at the level of individual protein molecules.

In a recent publication in Nature, Motone et al. report the development of a protein sequencing method using nanopores that enables the reading of long protein strands. This method allows for multi-pass re-reading and can detect single amino acid substitutions as well as post-translational modifications (PTMs).