Set-up for human sera MALDI profiling: the case of rhEPO treatment

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 even though the serum high dynamic range imposes some limitations, preventing detection and identification of less abundant species. Efforts to increase the MALDI profiling detection ability are needed. A set-up 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 spectra quality improvement. Immunodepletion skills of both columns were determined by 2D-DIGE, which precisely revealed the efficacy of Hu14 in protein removal and in serum dynamic range decrement. After optimization of the type of matrix and sample dilution, these new efficient conditions were used for serum profiling of ten healthy subjects before and after rhEPO treatment. The principal component analysis indicates that combination of Hu14 column and 2’,4’-dihydroxyacetophenone matrix increases data quality allowing to discriminate between treated and untreated samples, making serum MALDI profiling suitable for clinical monitoring of rhEPO.