Enhancing mass spectrometry based serum profiling by a combination of free flow electrophoresis and ClinProt™

Abstract

Pre-fractionation of serum specimen is a precondition for a reliable and sensitive proteomic analysis, but systemic variation of various techniques impairs the pre-analytical workflow and at least assay reproducibility. In the present study we introduce and evaluate a two-dimensional fractionation approach combining liquid-based isoelectric focusing (IEF) and affinity chromatography (ClinProt™ magnetic beads). In the first dimension serum specimen were fractionated according to the isoelectric point of protein components by free-flow electrophoresis (IEF-FFE). Subsequently, fractions were separated in a second dimension by ClinProt™ and protein profiling was done by MALDI-TOF analysis. Our investigation show that in contrast to ClinProt™ approach alone the combination of both techniques enhances detectable protein mass signals by a factor of fifteen coincided with high reproducibility (CV < 12% between different experiments). Therefore, we conclude that our combined approach improves the efficiency of MS based serum proteome analysis significantly.