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

作者：Bjarnadottir Stefania Guorun; Flengsrud Ragnar^*

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2014, 944: 107-113.

DOI：10.1016/j.jchromb.2013.11.004

• Affinity chromatography
• Fibrinogen
• Heparin-binding
• Biomarkers
• Immunodepletion

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.

• 出版日期2014-1-1