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

Francesca Lavatelli, Francesca Brambilla, Veronica Valentini, Paola Rognoni, Simona Casarini, Dario Di Silvestre, Vittorio Perfetti, Giovanni Palladini, Gabriele Sarais, Pierluigi Mauri, Giampaolo Merlini

Research output: Contribution to journalArticle

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: 98-2350 mg/L), 5 with ALκ amyloidosis and 1 with κ light chain (LC) myeloma (κ FLC range: 266-2660 mg/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.

Original languageEnglish
Pages (from-to)409-419
Number of pages11
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1814
Issue number3
DOIs
Publication statusPublished - Mar 2011

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Keywords

  • Amyloidosis
  • Immunoglobulin free light chains
  • Immunoprecipitation
  • Post-translational modifications
  • Proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Analytical Chemistry
  • Molecular Biology

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