title: Setup for human sera MALDI profiling: the case of rhEPO treatment.
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.
authors.affiliation: Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Milano, Milan, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Fania
authors.forename: Michele
authors.initials: M
authors.lastname: Vasso
authors.forename: Enrica
authors.initials: E
authors.lastname: Torretta
authors.forename: Paul
authors.initials: P
authors.lastname: Robach
authors.forename: Gaetano
authors.initials: G
authors.lastname: Cairo
authors.forename: Carsten
authors.initials: C
authors.lastname: Lundby
authors.forename: Cecilia
authors.initials: C
authors.lastname: Gelfi
mesh_terms: D001798:Blood Proteins / Q000302:isolation & purification*; D016903:Drug Monitoring; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000097:blood*; D006801:Humans; D007163:Immunosorbent Techniques / Q000295:instrumentation*; D008297:Male; D025341:Principal Component Analysis; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D001798:Blood Proteins; D011994:Recombinant Proteins; D004921:Erythropoietin