A novel approach for the purification and proteomic analysis of pathogenic immunoglobulin free light chains from serum.
Biochim Biophys Acta
; 1814(3): 409-19, 2011 Mar.
Article
em En
| MEDLINE
| ID: mdl-21215335
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.
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Bases de dados:
MEDLINE
Assunto principal:
Cadeias kappa de Imunoglobulina
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Cadeias lambda de Imunoglobulina
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Cadeias Leves de Imunoglobulina
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Proteômica
Limite:
Adult
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Aged
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Female
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Humans
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Male
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Middle aged
Idioma:
En
Revista:
Biochim Biophys Acta
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Itália