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

• Differences in the porcine and piscine heparin binding pattern were detected by 2D-electrophoresis.
• Seventeen fibrinogen isomers were identified as heparin-binding.
• Heparin chromatography and adapted immunodepletion resulted in the identification of 76 candidate biomarkers.
• Fourteen previously undescribed heparin-binding proteins were identified.
• Low abundance plasma proteins were identified.

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 × 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 × 75 μm)-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.