High-Coverage Stereoproteome Mapping Uncovers Pervasive Protein Stereoisomerization Associated with Neurodegeneration

Protein asymmetry, while crucial for life, can arise from subtle stereoisomerization. However, a comprehensive understanding of the breadth and specificity of the whole stereoproteome (STEP) has been hindered by insufficient stereoisomeric resolution. Here, we introduce an untargeted, <i>de novo</i> STEP discovery protocol for comprehensive STEP profiling and relative quantification. This method employs multidimensional isomeric separation, advanced algorithms, and stereoisomer-specific retention time shifts. STEP mapping identifies 182 neurodegenerative disease-linked, putative stereoisomeric proteins with stereoisomeric ratios of up to 70% in a cell model. A machine learning-derived scoring model achieves high confidence in endogenous stereoisomeric data assessment, achieving an average score of over 0.97 and a modeling accuracy exceeding 98%, with a false discovery rate of less than 5%. Validation experiments using synthetic STEP peptide standards and additional enzymatic localization of D-sites with aminopeptidase M confirm the putative STEP list and their relative abundances. This work advances protein stereoisomer analysis to a proteome scale, connecting protein molecular asymmetry with potential cellular functions and disease mechanisms.