Proteomics and Alkaloid Signature of Narrow Leaf Lupin Leaves Reveal Key Enzymes in Quinolizidine Alkaloid Biosynthesis and Transportation

Quinolizidine alkaloids (QAs) are toxic secondary metabolites in narrow-leafed lupin that defend against herbivores but constrain food and feed use. We developed a single-sample multiomics workflow for simultaneous extraction and quantitative analysis of QAs and proteins from individual leaves using urea/thiourea/CHAPS extraction with acetone precipitation, validated for QA quantification by LC-MS/MS. Data-independent acquisition proteomics of eight lupin accessions (four bitter, four sweet) identified 4437 proteins, with 183 differing significantly between bitter and sweet groups. Bitter accessions were enriched in lysine biosynthesis and stress-response pathways, whereas sweet accessions showed enrichment in phenylpropanoid and other defense-related processes. Coexpression network analysis linked protein modules to total and individual QA levels, capturing known QA-related proteins and revealing candidate enzymes and transporters. This workflow advances molecular understanding of QA regulation and provides candidate protein markers and targets to support breeding of low-alkaloid varieties for food and feed applications.