Single-molecule protein sequencing: One step closer to the ultimate performance of protein biomarker assays.

Single-molecule protein sequencing (SMPS) represents an innovative frontier in proteomics, emerging as a transformative approach to directly read individual protein molecules without the need for ensemble averaging. Unlike conventional methods for proteomic analysis, SMPS technologies offer ultimate analytical sensitivity for single-molecule detection and unparalleled analytical specificity for reading of amino acid sequences and post-translational modifications. This review summarizes the current landscape of SMPS technologies, emphasizing nanopore sequencing, degradation-based sequencing, and single-molecule mass spectrometry. Prospective applications of SMPS in clinical chemistry are highlighted. Challenges such as reproducibility and scalability are discussed alongside future perspectives.