title: A novel approach for the purification and proteomic analysis of pathogenic immunoglobulin free light chains from serum.
abstract: 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-κ or λ FLC antibodies. Isolated FLC were analyzed by SDS-PAGE, 2D-PAGE, immunoblotting, mass spectrometry (MS). Serum FLC were immunoprecipitated from 15 patients with ALλ amyloidosis (serum λ FLC range: 98-2350mg/L), 5 with ALκ amyloidosis and 1 with κ light chain (LC) myeloma (κ FLC range: 266-2660mg/L), and 3 controls. Monoclonal FLC were the prevalent eluted species in patients. On 2D-PAGE, both λ and κ FLC originated discrete spots with multiple pI 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.
authors.affiliation: Centro per lo Studio e la Cura delle Amiloidosi Sistemiche, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Lavatelli
authors.forename: Francesca
authors.initials: F
authors.lastname: Brambilla
authors.forename: Veronica
authors.initials: V
authors.lastname: Valentini
authors.forename: Paola
authors.initials: P
authors.lastname: Rognoni
authors.forename: Simona
authors.initials: S
authors.lastname: Casarini
authors.forename: Dario
authors.initials: D
authors.lastname: Di Silvestre
authors.forename: Vittorio
authors.initials: V
authors.lastname: Perfetti
authors.forename: Giovanni
authors.initials: G
authors.lastname: Palladini
authors.forename: Gabriele
authors.initials: G
authors.lastname: Sarais
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Mauri
authors.forename: Giampaolo
authors.initials: G
authors.lastname: Merlini
mesh_terms: D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000686:Amyloidosis / Q000097:blood; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000097:blood* / Q000302:isolation & purification; D007145:Immunoglobulin kappa-Chains / Q000302:isolation & purification*; D007146:Immunoglobulin lambda-Chains / Q000302:isolation & purification*; D047468:Immunoprecipitation / Q000379:methods; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D016415:Sequence Alignment
chemical_list: D000911:Antibodies, Monoclonal; D007147:Immunoglobulin Light Chains; D007145:Immunoglobulin kappa-Chains; D007146:Immunoglobulin lambda-Chains