Enhancing mass spectrometry based serum profiling by a combination of free flow electrophoresis and ClinProt.
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.
Hartwig
Sonja
S
Department of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center at the Heinrich-Heine-University Düsseldorf, Leibniz Center for Diabetes Research, Auf'm Hennekamp 65, Düsseldorf, Germany.
Kotzka
Jorg
J
Müller
Heidi
H
Müller-Wieland
Dirk
D
Eckel
Jürgen
J
Lehr
Stefan
S
D002846:Chromatography, Affinity / Q000379:methods*; D005260:Female; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D008297:Male; D013058:Mass Spectrometry; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D044967:Serum
D020543:Proteome