engleski

High-throughput fractionation of human plasma for fast enrichment of low- and high-abundance proteins

Methods were optimized to chromatographically isolate IgM from human plasma. Several parameters, including chromatography resin, mobile phases, and order of chromatographic separations performed were investigated and successful purification of IgM was ultimately achieved most successfully through isolation of immunoglobulin from human plasma using Protein A chromatography followed by subsequent fractionation using QA strong anion exchange chromatography. The level of purity of the isolated IgM, confirmed by SDS-PAGE and IgM- specific immunoblot, allows for further IgM- specific analysis of plasma samples. Through these optimization experiments, an additional method was established to deplete plasma of high-abundance proteins and decrease proteome complexity through fractionation sufficiently enough to perform successful label-free quantitative analysis of the plasma’s low abundance proteome with mass spectrometry. The developed fractionation scheme can be used for high-throughput screening of human plasma in order to identify low and high abundance proteins as potential prognostic and diagnostic disease biomarkers.

protein purification; high-throughput screening of human plasma; immunoglobulins; biomarkers

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