Proteomics as a tool for optimization of human plasma protein separation.
The application of proteomics technology in purification of proteins from human plasma and for characterization of plasma-derived therapeutics has been recently discussed. However, until now, the impact of this technology on the plasma protein fractionation and analysis of the final product has not been realized. In the present work, we demonstrate the use of proteomic techniques the monitoring of the first step of the plasma fractionation by use of anion-exchange chromatography. This chromatographic method is frequently used in the purification scheme for isolation of vitamin K dependent clotting factors II, VII, IX and X, and clotting inhibitors protein C and protein S, as well as inter-alpha inhibitor proteins (IaIp). After the removal of immunoglobulin G and non-binding proteins in the flow-through fraction, albumin and weakly bound proteins were eluted with low concentration of sodium chloride. The proteins that strongly bind to the anion-exchange column were eluted by higher salt concentrations. The fractions of interest were analyzed, and proteins were identified by LC-ESI-MS/MS. By use of this method, not only candidates for therapeutic concentrates, but also some potentially harmful components were identified. This strategy was very helpful for further process optimization, fast identification of target proteins with relatively low abundance, and for the design of subsequent steps in their removal or purification.
Kovac
Spomenka
S
J.J. Strossmayer University of Osijek, Department of Chemistry, HR-31000 Osijek, Croatia.
Yang
Xinli
X
Huang
Feilei
F
Hixson
Douglas
D
Josic
Djuro
D
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D001798:Blood Proteins