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, de novo 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.