Mass Spectrometry-Based De Novo Sequencing of N-Glycosylated Light Chains: Linking Urinary Proteoforms to In Situ Renal Amyloid Deposits
peer-reviewed · Molecular & Cellular Proteomics · 2026
| Date | 2026-09-01 |
| Type | peer-reviewed |
| Venue | Molecular & Cellular Proteomics |
| Publisher | Elsevier BV |
| Contribution | downstream-application |
| DOI | 10.1016/j.mcpro.2026.101652 |
| Citations (OpenAlex) | 0 |
| Venue 2-year citedness | 4.69 |
Abstract
Systemic 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.
Methods and tools
- AL amyloid light-chain proteoform sequencing: Pipeline for systemic AL amyloidosis that combines intact-mass Q-TOF measurement before and after deglycosylation with multi-protease digestion and de novo peptide sequencing, linking urinary N-glycosylated light-chain proteoforms to in situ renal amyloid deposits.
Cites (4)
- Decoding Protein Glycosylation by an Integrative Mass Spectrometry-Based De Novo Sequencing Strategy (2025) both
- Human plasma IgG1 repertoires are simple, unique, and dynamic (2021) both
- De Novo MS/MS Sequencing of Native Human Antibodies (2017) both
- Novor: Real-Time Peptide de Novo Sequencing Software (2015) both