iProXProteomicsSuxia WangHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0016431000suxiawang@bjmu.edu.cnPeking University First HospitaliProXSystemic immunoglobulin light-chain (AL) amyloidosis is characterized by clonal immunoglobulin-secreting cells that produce a monoclonal light chain prone to misfolding and amyloid fibril formation in tissues. Understanding its molecular basis requires accurate full-length sequencing of amyloidogenic light chains and linkage of circulating light chains to renal deposits. Achieving this is technically challenging because the low abundance and N-glycosylation of amyloidogenic light chains can complicate protein purification and peptide-level sequence analysis. To address these challenges, we implemented a robust analytical pipeline that integrates intact-mass measurement by Q-TOF MS before and after deglycosylation with multi-protease digestion and de novo peptide sequencing to systematically characterize urinary N-glycosylated light chains. Mass shifts observed before and after deglycosylation supported the presence of N-glycosylation, whereas deglycosylated intact masses were used to constrain full-length sequence assembly. The assembled urinary light-chain sequences were subsequently compared with matched renal amyloid proteomes isolated by laser microdissection and analyzed by bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS). Enzymatic deglycosylation reduced glycan-induced spectral interference. Eight monoclonal light-chain sequences were assembled from urinary light chains, including five derived from the immunoglobulin kappa variable 1 (IGKV1), two from the immunoglobulin lambda variable 2 (IGLV2), and one from IGKV4. The close agreement between theoretical and experimental intact masses confirmed the accuracy and completeness of sequence assembly. In each renal amyloid proteome, the corresponding urine-derived sequence had the highest score among light-chain identifications of the patient's clinically determined isotype (κ or λ) and showed 87.6% to 100% variable-region peptide coverage. Together, these findings support urinary N-glycosylated monoclonal light chains as the precursor proteins of the corresponding renal amyloid fibrils. In conclusion, we have demonstrated a robust workflow that applies established de novo peptide sequencing to characterize urinary N-glycosylated light chains and trace the corresponding sequences in renal amyloid deposits, with potential applications across light chain-related diseases.Mass Spectrometry-Based De Novo Sequencing of N-Glycosylated Light Chains: Linking Urinary Proteoforms to In Situ Renal Amyloid Deposits.Zhu Yueyue Y, Wang Xin X, Zhou Xinyue X, Wang Shuang S, Ji Peifeng P, Yan Zidi Z, Chen Kai K, Yu Xiaojuan X, Liu Gang G, Yang Li L, Liu Yi Y, Wang Suxia SfalseDe Novo Sequencing of N-Glycosylated Light Chains in AL AmyloidosisWe established an integrative de novo sequencing workflow to resolve the complete amino acid sequence of N-glycosylated LCs isolated from the urine of AL patients.Thu Apr 16 00:00:00 GMT+01:00 2026PXD077287960642680105