title: Affinity chromatography, two-dimensional electrophoresis, adapted immunodepletion and mass spectrometry used for detection of porcine and piscine heparin-binding human plasma proteins.
abstract: Heparin-binding proteins in human plasma were studied using affinity chromatography columns with porcine (2mL, 10.7mg capacity) and piscine heparin (5mL, 2.7mg capacity). Two-dimensional electrophoresis (Bio-Rad Protean II gel system with 16cm×16cm gels using isoelectric focusing (IEF) and nonequilibrium pH-gradient gel electrophoresis (NEPHGE)), Bruker Ultraflex MALDI-TOF mass spectrometry and immunoblotting (NovaBlot semidry discontinuous blotting) were used for unfractionated plasma. This revealed electropherograms with differences between porcine and piscine heparin-binding and totally 17 different fibrinogen variants from all 3 chains. Immunodepletion was used to remove fibrinogen (42.1mg anti-human fibrinogen in 8.4mL resin) and serum albumin (0.42mg binding capacity in 14mL resin) and porcine and piscine heparin-binding proteins were identified using liquid chromatography-mass spectrometry (Ultimate 3000 NanoLC with Acclaim PepMap 100 column (50cm×75μm)-LTQ Orbitrap Mass XL). In total, the binding of 76 putative or acknowledged biomarkers are shown. Of the identified proteins, 14 are not previously shown to be heparin-binding, such as the low concentration proteins lipocalin-1 and tropomyosin and a hitherto not detected protein in plasma, zinc finger protein 483. The putative heparin-binding sequences were analyzed. The results suggest that the combination of group specific affinity and adapted immunodepletion chromatography could be useful in the study of the plasma proteome.
authors: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
authors: Stefanía Guðrún
authors: SG
authors: Bjarnadóttir
authors: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway. Electronic address: ragnar.flengsrud@umb.no.
authors: Ragnar
authors: R
authors: Flengsrud
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D029941:Fish Proteins / Q000737:chemistry / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D007163:Immunosorbent Techniques; D013058:Mass Spectrometry; D020317:Salmo salar; D013552:Swine
chemical_list: D001798:Blood Proteins; D029941:Fish Proteins; D055767:Immobilized Proteins; D006493:Heparin
keywords: Affinity chromatography; Biomarkers; Fibrinogen; Heparin-binding; Immunodepletion