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2-microglobulin is not detectable by 2D electrophoresis in ex vivo amyloid fibrils of two patients affected by dialysis-related amyloidosis
1 Department of Biochemistry, University of Pavia-Laboratori di Biotecnologie IRCCS Policlinico San Matteo, 27100 Pavia, Italy
2 Centre for Amyloidosis and Acute Phase Proteins, Department of Medicine, Royal Free and University College Medical School, University College London, London NW3 2PF, United Kingdom
3 Department of Physics, University of Genoa, 16146 Genoa, Italy
4 CEINGE Biotecnologie Avanzate, scarl, Università di Napoli Federico II, 80145 Napoli, Italy
5 Department of Autoimmunology, Statens Serum Institute, DK-2300 Copenhagen S, Denmark
6 Department of Biomedical Science and Technology, University of Udine, 33100 Udine, Italy
(RECEIVED September 15, 2006; FINAL REVISION October 21, 2006; ACCEPTED November 1, 2006)
The lysine 58 cleaved and truncated variant of 
2-microglobulin (
K58-2m) is conformationally unstable and present in the circulation of a large percentage of patients on chronic hemodialysis, suggesting that it could play a role in the 2-microglobulin (2m) amyloid fibrillogenesis associated with dialysis-related amyloidosis (DRA). However, it has yet to be detected in the amyloid deposits of such patients. Here, we extracted amyloid fibrils, without denaturation or additional purification, from different amyloidotic tissues of two unrelated individuals suffering from DRA, and characterized them by high-sensitivity bidimensional gel electrophoresis (2D-PAGE), immunoblotting, MALDI time-of-flight mass spectrometry, and protein sequencing. To confirm whether or not this species could be identified by our proteomic approaches, we mapped its location in 2D-PAGE, in mixtures of pure K58-2m, and extracts of amyloid fibrils from patients, to a discrete region of the gel distinct from other isoforms of 2m. Using this approach, the two known principal isoforms found in 2m amyloid were identified, namely, the full-length protein and the truncated species lacking six N-terminal amino acid residues (N6-2m). In contrast, we found no evidence for the presence of K58-2m.
Keywords: dialysis related amyloidosis; amyloid fibrils; proteolized variants of 2-microglobulin; proteomics
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