Peptide-level assay of protein interaction using combinatorial native and denatured proteolysis

PLAPI of a single human serum specimen identified 124 peptides detected under both native and denatured proteolysis, including 13 denatured-only C3b peptides at the Factor B binding interface.

• Why it matters: Structural interfaces in endogenous proteomes are hard to pinpoint from complex samples, and peptide recovery changes with condition need complementary mapping to structural regions.
• What they did: The team performed brief trypsin digestion under native conditions and extended digestion after denaturation in three paired technical preparations, then used isotopic diethyl labeling with LCMS/MS and a 2 of 3 detection rule.
• The result: Mapping to reference structures linked denatured-only peptides to specific interface regions, and STRING association plus enrichment connected candidate proteins to extracellular defense and protease regulation, enabling peptide-level prioritization for follow-up.

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