The implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. MALDI profiling is a robust and sensitive technology although the serum high dynamic range imposes a major limitation hampering the identification of less abundant species decreasing the quality of MALDI profiling. A setup to improve these parameters has been performed for recombinant human erythropoietin (rhEPO) monitoring in serum, analyzing the effects of two commercially available columns (MARS Hu7 and Hu14) for immunodepletion, and two matrices (α-cyano-4-hydroxycinnamic acid and 2′,4′-dihydroxyacetophenone) for peak quality improvement. The immunodepletion capability of both columns was determined by 2-D DIGE, which precisely revealed the efficacy of Hu14 in protein removal and the serum dynamic range decrement. In addition, the type of matrix, the sample dilution, and the efficacy of optimized parameters were used for serum profiling of ten healthy subjects before and after rhEPO treatment. The principal component analysis indicates that a combination of Hu14 column and 2′,4′-dihydroxyacetophenone matrix increases data quality allowing the discrimination between treated and untreated samples, making serum MALDI profiling suitable for clinical monitoring of rhEPO

Setup for human sera MALDI profiling: The case of rhEPO treatment / C. Fania, M. Vasso, E. Torretta, P. Robach, G. Cairo, C. Lundby, C. Gelfi. - In: ELECTROPHORESIS. - ISSN 0173-0835. - 32:13(2011), pp. 1715-1727. [10.1002/elps.201100134]

Setup for human sera MALDI profiling: The case of rhEPO treatment

C. Fania
Primo
;
M. Vasso
Secondo
;
E. Torretta;G. Cairo;C. Gelfi
Ultimo
2011

Abstract

The implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. MALDI profiling is a robust and sensitive technology although the serum high dynamic range imposes a major limitation hampering the identification of less abundant species decreasing the quality of MALDI profiling. A setup to improve these parameters has been performed for recombinant human erythropoietin (rhEPO) monitoring in serum, analyzing the effects of two commercially available columns (MARS Hu7 and Hu14) for immunodepletion, and two matrices (α-cyano-4-hydroxycinnamic acid and 2′,4′-dihydroxyacetophenone) for peak quality improvement. The immunodepletion capability of both columns was determined by 2-D DIGE, which precisely revealed the efficacy of Hu14 in protein removal and the serum dynamic range decrement. In addition, the type of matrix, the sample dilution, and the efficacy of optimized parameters were used for serum profiling of ten healthy subjects before and after rhEPO treatment. The principal component analysis indicates that a combination of Hu14 column and 2′,4′-dihydroxyacetophenone matrix increases data quality allowing the discrimination between treated and untreated samples, making serum MALDI profiling suitable for clinical monitoring of rhEPO
2-D differential gel electrophoresis; EPO; Immunodepletion; MALDI profiling; Serum
Settore MED/04 - Patologia Generale
Settore BIO/12 - Biochimica Clinica e Biologia Molecolare Clinica
   Identifying and validating pre-clinical biomarkers for diagnostics and therapeurtics of Neuromuscolar Disorders
   BIO-NMD
   EUROPEAN COMMISSION
   FP7
   241665
2011
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/159024
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