Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode

Abstract

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately 20 years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low-abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS.

Figure 1

Separation of plasma proteins by HIC. After treatment with 1.7 M (NH₄)₂SO₄ in 10 mM Tris-HCl, pH 7.4 (Buffer A) and removal of precipitated proteins by centrifugation (see Materials and methods) 26 mg of plasma protein from the supernatant were applied to the Toyopearl Phenyl 650 S column (I.D. 6.5 mL, column volume 0.34 mL). After washing with 5 mL of Buffer A, bound proteins were eluted with 10 mM Tris-HCl, pH 7.4 (Buffer B). A – Sample application step; B – elution step. Optical density was determined at 280, 260 and 210 nm. Chromatographic conditions – Flow rate 0.5 mL/min, temperature 4–8°C, pressure 1 bar. Collected fractions were analyzed by SDS-PAGE, and LC-MS/MS – see Table 1A.

Figure 1

Separation of plasma proteins by HIC. After treatment with 1.7 M (NH₄)₂SO₄ in 10 mM Tris-HCl, pH 7.4 (Buffer A) and removal of precipitated proteins by centrifugation (see Materials and methods) 26 mg of plasma protein from the supernatant were applied to the Toyopearl Phenyl 650 S column (I.D. 6.5 mL, column volume 0.34 mL). After washing with 5 mL of Buffer A, bound proteins were eluted with 10 mM Tris-HCl, pH 7.4 (Buffer B). A – Sample application step; B – elution step. Optical density was determined at 280, 260 and 210 nm. Chromatographic conditions – Flow rate 0.5 mL/min, temperature 4–8°C, pressure 1 bar. Collected fractions were analyzed by SDS-PAGE, and LC-MS/MS – see Table 1A.

Figure 2

Separation of proteins from human plasma on the hydrophobic interaction support Toyopearl Phenyl 650 S (column volume 0.34 mL). 1.7 M (NH₄)₂SO₄ in 10 mM Tris-HCl, pH 7.4, was used as Buffer A. Increasing amount of human plasma was applied. Bound proteins were eluted with 10 mM Tris-HCl, pH 7.4. SDS-PAGE of eluted proteins is shown. For other conditions – see Materials and methods and Figure 1.

Figure 3

Separation of proteins from human plasma on the hydrophobic interaction support Toyopearl Phenyl 650 S (column volume 0.34 mL). 4 M NaCl in 10 mM Tris-HCl, pH 7.4 was used as Buffer A. Increasing amount of human plasma was applied. Bound proteins were eluted with 10 mM Tris-HCl, pH 7.4. SDS-PAGE of eluted proteins is shown. For other conditions – see Materials and methods and Figure 1.

Figure 4

Sample displacement chromatography of plasma proteins by use of three identical columns packed with Toyopearl Phenyl 650 S (column volume 0.34 mL each). A – all three columns were connected in series during sample loading and washing. B – for the elution step, the columns were detached and bound proteins were eluted by a step gradient of 100% Buffer B (10 mM Tris-HCl, pH 7.4). Different grades of yellow color express the different relative concentration of serum albumin, which carries yellow colored pigment from human plasma.

Figure 4

Sample displacement chromatography of plasma proteins by use of three identical columns packed with Toyopearl Phenyl 650 S (column volume 0.34 mL each). A – all three columns were connected in series during sample loading and washing. B – for the elution step, the columns were detached and bound proteins were eluted by a step gradient of 100% Buffer B (10 mM Tris-HCl, pH 7.4). Different grades of yellow color express the different relative concentration of serum albumin, which carries yellow colored pigment from human plasma.

Figure 5

Sample displacement chromatography of plasma proteins by use of three identical columns packed with Toyopearl Phenyl 650 S (column volume 0.34 mL each). SDS-PAGE of proteins eluted from each column after detachment (see Figure 4 and Materials and methods). A - 1.7 M (NH₄)₂SO₄ in 10 mM Tris-HCl, pH 7.4 was used as Buffer A; B - 4 M NaCl in 10 mM Tris-HCl, pH 7.4 was used during sample application and washing. For other conditions – see Figure 4.

Figure 5

Sample displacement chromatography of plasma proteins by use of three identical columns packed with Toyopearl Phenyl 650 S (column volume 0.34 mL each). SDS-PAGE of proteins eluted from each column after detachment (see Figure 4 and Materials and methods). A - 1.7 M (NH₄)₂SO₄ in 10 mM Tris-HCl, pH 7.4 was used as Buffer A; B - 4 M NaCl in 10 mM Tris-HCl, pH 7.4 was used during sample application and washing. For other conditions – see Figure 4.