A novel approach for the purification and proteomic analysis of pathogenic immunoglobulin free light chains from serum

An excess of circulating monoclonal free immunoglobulin light chains (FLC) is common in plasma cell disorders. A subset of FLC, as amyloidogenic ones, possess intrinsic pathogenicity. Because of their complex purification, little is known on the biochemical features of serum FLC, possibly related to their pathogenic spectrum. We developed an immunopurification approach to isolate serum FLC from patients with monoclonal gammopathies, followed by proteomic characterization. Serum monoclonal FLC were detected and quantified by immunofixation and immunonephelometry. Immunoprecipitation was performed by serum incubation with agarose beads covalently linked to polyclonal anti-kappa or lambda FLC antibodies. Isolated FLC were analyzed by SDS-PAGE, 20-PAGE, immunoblotting, mass spectrometry (MS). Serum FLC were immunoprecipitated from 15 patients with AL lambda amyloidosis (serum lambda FLC range: 98-2350 mg/L), 5 with AL kappa amyloidosis and 1 with kappa light chain (LC) myeloma (K FLC range: 266-2660 mg/L, and 3 controls. Monoclonal FLC were the prevalent eluted species in patients. On 2D-PAGE, both lambda and kappa FLC originated discrete spots with multiple pl isoforms. The nature of eluted FLC and coincidence with the LC sequence from the bone marrow clone was confirmed by MS, which also detected post-translational modifications, including truncation, tryptophan oxidation, cysteinylation, peptide dimerization. Serum FLC were purified in soluble form and adequate amounts for proteomics, which allowed studying primary sequence and detecting post-translational modifications. This method is a novel instrument for studying the molecular bases of FLC pathogenicity. allowing for the first time the punctual biochemical description of the circulating forms. (c) 2011 Elsevier B.V. All rights reserved.