Affinity chromatography, two-dimensional electrophoresis, adapted immunodepletion and mass spectrometry used for detection of porcine and piscine heparin-binding human plasma proteins

Heparin-binding proteins in human plasma were studied using affinity chromatography columns with porcine (2 mL, 10.7 mg capacity) and piscine heparin (5 mL, 2.7 mg capacity). Two-dimensional electrophoresis (Bio-Rad Protean II gel system with 16 cm x 16 cm 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 (42.1 mg anti-human fibrinogen in 8.4 mL resin) and serum albumin (0.42 mg binding capacity in 14 mL resin) and porcine and piscine heparin-binding proteins were identified using liquid chromatography-mass spectrometry (Ultimate 3000 NanoLC with Acclaim PepMap 100 column (50 cm x 75 mu m)-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. (C) 2013 Elsevier B.V. All rights reserved.