Dye Affinity Chromatography

Affinity Chromatography

D.S. Hage , ... X. Zheng , in Liquid Chromatography: Applications, 2013

1.5 Dye-Ligand and Biomimetic Affinity Chromatography

Synthetic dyes and chlorotriazine-linked biomimetic ligands are another group of ligands that can be used in affinity chromatography. These ligands have been used in numerous applications to purify enzymes and proteins. Dyes and biomimetic ligands are easy to immobilize, inexpensive, stable, and provide stationary phases with high binding capacities. These features make such ligands of interest in large-scale or high-throughput separation techniques for the development of protein-based drugs or the examination of protein libraries [50–54]. Dye-ligand affinity chromatography often uses triazine dyes to purify albumin and other blood proteins, as well as enzymes and pharmaceutical proteins [50,52,54]. Figure 1.3 shows some typical dye ligands used in dye-affinity chromatography, including Cibracron Blue 3GA and Procion Blue. These dyes have two units joined through an amino-bridge. The first unit usually contains an anthraquinone group but can also contain an azo or phthalocyanine group. The second unit usually contains a triazine and forms a scaffold for the binding domain and groups that can be used to attach the ligand to a support [50,53,55]. These dye-ligands often have negatively charged sulfonic groups, which gives them some cation-exchange properties [52,56]. Retained proteins may be dissociated from these columns by using nonspecific elution; however, biospecific elution through the addition of a competing agent to the mobile phase is usually the preferred approach [52,57].

FIGURE 1.3. Structures of Cibacron Blue 3GA and Procion Blue, two common binding agents used in dye-ligand affinity chromatography.

 [52]

To increase the speed and efficiency of dye-ligand purifications, a method known as polymer-shielded dye-affinity chromatography is sometimes employed [58,59]. In this technique, the stationary phase is treated with a water-soluble polymer to prevent nonspecific interactions between proteins and the dye. This method has been effectively used in the purification of enzymes [59]. Other applications of dye ligands include their use in removing toxic macromolecules from biological fluids, such as prion proteins, human immunodeficiency virus-1, and hepatitis B particles [60–62]. There is also growing interest in using computational and combinatorial chemistry, in a method known as biomimetic affinity chromatography, to develop improved dye-based ligands and related binding agents for use in the purification of pharmaceutical proteins [50].

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Protein Liquid Chromatography

Jürgen Kirchberger , Hans-Joachim Böhme , in Journal of Chromatography Library, 2000

8.4. Application Examples

8.4.1. Purification of alkaline phosphatase from calf intestine

A representative protocol employing affinity partitioning in aqueous two-phase systems as ligand screening method and the development of an efficient purification procedure for alkaline phosphatase from calf intestine is described [27].

8.4.1.1. Screening of dye-ligands

The effect of dye-ligands on the partitioning of purified alkaline phosphatase was studied by measuring the partition coefficient of the enzyme in the presence of various dyes bound to PEG 6000. The partition coefficient K is defined as the activity ratio of the enzyme in the top and bottom phase. The difference (ΔlogK) in the log K values obtained from samples with and without dye-liganded PEG is regarded as a measure of the affinity partitioning effect (extraction power).

Systems (4 g) composed of 6.5% (w/w) PEG 6000 and 9.75% (w/w) dextran M 70, showing a logK of –0.8 and a volume ratio between the top and bottom phase of 1.24 were selected for studying the affinity partition effect. The amount of dye-liganded PEG was kept constant at 5% of the total PEG which yields in most cases maximum extraction (ΔlogK max). In this example a purified enzyme (2200 U/mg) was used, although this method is also applicable for crude extracts.

Ten units (10 μl) of the dialyzed enzyme were added to the systems at 4°C. After equilibration by gentle mixing for 20 s, the systems were centrifuged at 2500 × g for 2 min and the alkaline phosphatase activity measured in the top and bottom phase.

Fig. 8.2 shows the ΔlogK values determined for 41 different dye-ligands. Only with Procion Navy H-ER (Blue 171), Procion Red H-E3B (Red 120) and Procion Yellow H-E3G (Yellow 81) ΔlogK values greater than 1.4 were obtained. In order to determine the relative affinity (0.5 × ΔlogK max) and the maximum extraction power (ΔlogK max) the dependence of ΔlogK on the concentration of these three dyes in the systems was determined (Fig. 8.3). By using the double reciprocal plots of the ΔlogK versus the dye-PEG concentration (data not shown) it became significant that even then all three dyes generated nearly the same maximum extraction power. Procion Navy H-ER possessed the highest relative affinity and was therefore chosen as the ligand in dye-affinity chromatography of alkaline phosphatase from calf intestine. Furthermore, it is shown, that the competitive inhibitor inorganic phosphate abolishes the affinity partitioning effect (Fig. 8.3) and might be a useful eluant for the affinity chromatography of alkaline phosphatase on matrix-bound Procion Navy H-ER.

Fig. 8.2. Effect of different dye-PEG derivatives on the affinity partitioning of alkaline phosphatase. The systems (4 g) were composed of 9.75% (w/w) dextran M 70 and 6.5% (w/w) PEG 6000 including 5% of the respective dye-PEG derivative, 10 mM Tris•HCl buffer, pH 7.5, 2 mM MgCl2 and 10 units of purified enzyme. The systems were equilibrated at 4°C. The columns are labelled according the Colour Index (generic name) of the dyes.

Fig. 8.3. Influence of the concentrations of dye-PEG and inorganic phosphate on the affinity partitioning effect (ΔlogK) of alkaline phosphatase. The systems (4 g) contained 9.75% (w/w) dextran M 70, 6.5% (w/w) PEG 6000, 10 mM Tris•HCl buffer, pH 7.5, 2 mM MgCl2 and 10 units of purified enzyme. All manipulations were done at 4°C. The insert shows the influence of inorganic phosphate on the partitioning of alkaline phosphatase in a Reactive Blue 181-PEG (1% w/w) containing an aqueous two-phase system. (•) Procion Navy H-ER (Reactive Blue 181); (▴) Procion Red H-E3B (Reactive Red 120); (♦) Procion Yellow H-E3G (Reactive Yellow 81).
8.4.1.2. Purification procedure

The enzyme was solubilized from duodenal calf intestine by using butanol extraction and homogenization. The cell debris was removed by applying aqueous phase partitioning in systems composed of 10% (w/w) PEG 4000, 5% dextran (M r: 50 000), 75% (w/w) extract in 10 mM Tris•HCl-buffer, pH 7.5, 2 mM MgCl2 (buffer A) at 4°C. The top phase was diluted with water and mixed with DEAE-cellulose in 10 ml Tris•HCl-buffer, pH 8.0, 2 mM MgCl2 (buffer B) at 4°C. After binding and washing in a batch procedure the enzyme was eluted with 50 mM NaCl in buffer B and concentrated by ultrafiltration.

The concentrated sample was dialyzed exhaustively against buffer A and loaded onto a column of Procion Navy H-ER-Sepharose 4B equilibrated with buffer A at 4°C. For 350 mg of enzyme with a specific activity of about 100 units/mg, 100 ml of dye-liganded gel were used. After washing the column, the enzyme was eluted with 5 mM potassium phosphate buffer. A typical chromatogram is presented in Fig. 8.4. Enzyme-containing fractions were pooled and concentrated by ultrafiltration, giving an overall yield of 59% and a specific activity of 2200 U/mg. The enzyme was homogeneous as judged by PAGE (Fig. 8.5) and could be stored in 30 mM triethanolamine·HCl buffer, 3 M NaCl, 1 mM MgCI2, 0.1 mM ZnCl2, at 4°C without loss of activity for at least 6 months. Table 8.7 summarizes a typical purification procedure.

Fig. 8.4. Dye-ligand affinity chromatography of alkaline phosphatase. The Procion Navy H-ER-Sepharose 4B column (1.5 × 8 cm) was equilibrated with 10 mM Tris•HCl buffer, pH 7.5, 2 mM MgCl2 (buffer A). The dialyzed partial purified enzyme (4900 units) (100 U/mg) was applied. The column was washed with 70 mM NaCl in buffer A until the eluate was free of protein (A 280m < 0.02). After removing the sodium chloride by washing with one volume of buffer A, the enzyme was eluted with 5 mM potassium phosphate in buffer A. The chromatography was performed at 4°C with a flow rate of 40 ml/h.

Fig. 8.5. Native PAGE of alkaline phosphatase from calf intestine. Electrophoresis was performed under non-denaturating conditions (3–15%, w/v, acrylamide) and the gel was stained with Coomassie Brillant Blue G 250. Lane 1: aqueous phase after butanol extraction; lane 2: top phase of the aqueous two-phase system; lane 3: DEAE-chromatography; lane 4: Procion Navy H-ER-Sepharose 4B.

Table 8.7. SCHEME OF THE PURIFICATION OF ALKALINE PHOSPHATASE FROM CALF INTESTINE

Purification step Total activity (U) Specific activity (U/mg) Purification (-fold) Recovery (%)
Homogenate 4450 1 1 80
Butanol extraction 5570 5 5 100
Aqueous two-phase partitioning 4900 10 10 88
DEAE-cellulose 3788 100 100 68
Procion Navy H-ER-Sepharose 3286 2200 2200 59

8.4.2. Purification of aldolase B from human liver

In this example, screening for the most suitable dye ligand was done kinetically [28]. As demonstrated in Table 8.8, the dyes investigated inhibit the enzyme in the micromolar range competitively with respect to the substrate fructose 1-phosphate. As Cibacron Blue F3G-A turned out to be the most effective inhibitor of the enzyme, this dye was used as ligand in dye-affinity chromatography of liver aldolase.

Table 8.8. INFLUENCE OF REACTIVE DYES ON THE KINETIC PARAMETERS OF HUMAN LIVER ALDOLASE

Reactive dye kcat (units/mg) K M (mM) K, (μM)
None 1.05 ± 0.02 2.59 ± 0.71 −
Cibacron Blue F3G-A 1.09 ± 0.02 0.91 ± 0.26 0.26 ± 0.06
Cibacron Brilliantblue FBR-P 0.96 ± 0.02 1.75 ± 0.15 1.18 ± 0.14
Procion Blue MX-R 0.98 ± 0.02 1.70 ± 0.08 7.52 ± 0.85
Procion Red H-E3B 1.15 ± 0.02 2.80 ± 0.21 0.82 ± 0.01
Procion Red H-3B 1.11 ± 0.02 2.64 ± 0.23 1.22 ± 0.15

Liver tissue was homogenized in two volumes of 50 mM Tris•HCl buffer, pH 7.5 containing 1 mM 2-mercaptoethanol and 5 mM EDTA. After centrifugation for 30 min at 9000 × g, the enzyme was fractionated with PEG 6000 (3–12%, w/v) and the pellet dissolved in 20 mM sodium phosphate, 2 mM EDTA and 1 mM 2-mercaptoethanol, pH 6.0. The solution was clarified by centrifugation and the enzyme solution mixed with CM-Sephadex C50 equilibrated with the same buffer. The ion exchanger was separated on a Buchner funnel and washed with buffer until the protein concentration in the effluent was negligible. After elution with 10 mM fructose 1,6-bisphosphate, the enzyme was concentrated by ultrafiltration and applied to a Blue Sepharose CL-6B column equilibrated with 10 mM Tris•HCl, 1mM EDTA, pH 8.5. The column was washed with the same buffer and the enzyme eluted with 1 mM fructose 1,6-bisphosphate (Fig. 8.6). The purified enzyme exhibits a single band in SDS-PAGE and has a specific activity of about 1.1 unit/mg. The overall yield is about 35–40%. Table 8.9 shows a representative protocol of this purification procedure.

Fig. 8.6. Chromatography of human liver aldolase on Blue Sepharose. The column was equilibrated with 10 mM Tris•HCl, 2-mercaptoethanol pH 8.5. About 9500 units of the concentrated eluate from the CM-Sephadex batch chromatography step were applied and the column was washed exhaustively with the same buffer. The enzyme was eluted by adding 1 mM fructose 1,6-bisphosphate to the buffer.

Table 8.9. PURIFICATION OF ALDOLASE B FROM HUMAN LIVER

Purification step Total activity (U) Specific activity (U/mg) Purification (-fold) Recovery (%)
Homogenate 16970 0.01 1 100
PEG precipitate 10920 0.02 2 64.4
CM-Sephadex 9440 0.12 12 55.6
Blue Sepharose 6450 1.10 110 38.0

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Applications of Amphiphilic Copolymers in Separations

Mårten Svensson , ... Folke Tjerneld , in Amphiphilic Block Copolymers, 2000

4 CHROMATOGRAPHY

4.1 Displacement and shielding chromatography

In affinity chromatography a ligand is covalently attached to a column matrix. When a sample of proteins containing a target protein is added to the column, a high purification can be obtained if only the target protein binds to the ligand. After washing the column, which removes unbound proteins, the target protein is eluted from the column by adding a soluble ligand to the column (specific elution) or by increasing ionic strength (non-specific elution). Strong binding between the ligand and target protein is desirable and this is can be obtained by choosing ligands with specific affinity to the target protein. However, specific ligands are usually expensive. The relatively inexpensive triazine dyes have been much used as ligands in affinity chromatography [56]. Nucleotide binding proteins have a strong affinity for these dyes. However, many proteins may also bind non-specifically to the ligand with an intermediate binding strength, which leads to a poor purification [57].

Shielding chromatography is a method to avoid that proteins with a weak or intermediate affinity to the ligand will bind to the ligand in the column. This is accomplished by preloading a column with a polymer, which binds to the ligand. When a sample of proteins is loaded on the column only the proteins with a very strong affinity to the ligand are bound, since the polymer is a competitor to the proteins for binding the ligands. The target protein which has a strong affinity to the ligand can compete with the polymer and will locally displace a polymer segment from binding to a ligand. This will decrease the total binding force between polymer and ligand but the polymer will normally not be eluted from the column since it can bind several ligands at the same time. The principle is shown in Figure 17.

Fig. 17. Shielding chromatography. (A) Loading the polymer shielded column with proteins. (B) Proteins with insufficient affinity for the ligand are washed out of the column. The polymer remains non-covalently bound to the column.

Shielding chromatography has been studied and developed by Galaev et al [57-58]. A strategy for the design of polymer shielding dye-affinity chromatography has been presented in ref. [59]. The chosen polymer should form a strong polymer-ligand complex, otherwise the proteins, target protein or added soluble ligands will displace the polymer, leading to polymer leaching. However, the polymer-ligand complex should not be too strong, which could prevent target proteins from binding. The strength of the polymer ligand complex can be modulated by changing polymer size. Larger polymers will bind stronger due to the numerous multi-point contacts with the ligand [59]. The shielding chromatography has been applied to the purification of lactate dehydrogenase (LDH) from porcine muscle. Polyvinylpyrrolidone (PVP-10) with molecular weight of 10 000 was used as shielding polymer in a Procion Blue HERD coupled sepharose column. Shielding was obtained by first percolating the column with 1% solution of PVP. LDH was eluted from the column with 1.5   M KCl. Finally, PVP was removed from the column by addition of a ligand, Cibacron Blue. Another method was to displace PVP with another polymer, polyethyleneimine (PEI). The LDH was purified 30-fold and with 94% recovery [59].

Displacement chromatography is similar to shielding chromatography [60]. The difference is that in the former, a polymer with much higher affinity for the ligand than the target protein, is added to the column after the target protein has been bound to the column. This leads to an effective elution of the target protein. The cationic PEI has been used to displace LDH from the negatively charged Cibacron Blue 3GA ligand (CB). In order to regenerate the column, PEI was removed by increasing the pH to 12. The reduced electrostatic attraction between PEI and CB, due to decreased charge of PEI, was further reduced with high ionic strength. This method was insufficient for a system with Procion Red HE-3B (PR) as ligand. In this system PEI was removed from the PR-bound column by including polyacrylic acid (PAA) in the high pH-high salt buffer. The highly charged PAA formed a soluble polyelectrolyte complex with the weakly charged PEI, which was thus eluted [60].

A new method which combines shielding and displacement effects has been developed by Galaev et al. [61]. In this method a thermoprecipitating polymer, polyvinylcaprolactam (PVCL) was used as a shielding polymer. This polymer thermoprecipitates at 38   °C (the cloud point temperature). In this system the target protein, LDH was bound to the column when the temperature was 40   °C (and ionic strength 0.1   M KCl). Upon decreasing the temperature below the cloud point, the LDH was eluted from the column at the same ionic strength, 0.1   M KCl. This effect was explained as follows: above the cloud point the polymer adopts a compact structure. This leads to a reduced strength of the polymer-ligand complex due to few multi-point contacts between the polymer and the ligands. This reduced affinity allows the LDH to compete with the polymer for ligand binding. Below the cloud point the polymer adopts a coil structure with more ligand binding as consequence. Thus the target protein cannot compete with the polymer and is eluted from the column.

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Protein and peptide separations

Uroš Andjelković , ... Djuro Josić , in Liquid Chromatography (Second Edition), 2017

5.2.2 Ion-Exchange Chromatography

Ion-exchange chromatography (IEX) has been used for more than 50 years for the separation and purification of proteins. In comparison with other chromatographic methods, 40% of all protein separations are related to IEX, 18% to SEC, and 29% to affinity chromatography (including immobilized metal-affinity and dye-affinity chromatography) [1].

IEX is a method for the purification of proteins based on ionic interactions between proteins and surface charges opposite to those of the charged groups on an ionic resin (see Fig. 5.3). In cation-exchange chromatography, positively charged molecules are attracted to a negatively charged solid support. Conversely, in anion-exchange chromatography, negatively charged molecules are attracted to a positively charged solid support. The separation occurs because of competition between proteins with different charges on an ion-exchange resin.

Fig. 5.3. Principles of ion-exchange chromatography (IEX). Biomolecules exhibit different degrees of interaction with charged chromatography media according to differences in their overall charge, charge density, and surface charge distribution. The separation process is based on the formation of ionic bonds between the charged groups on biomolecules (mostly, –NH3 +, =   NH2 +, ≡   NH+, –COO–, PO4 3–, and SO3 2–), and an ion-exchange support with the opposite charge. Nonbound biomolecules (i.e., neutral molecules with no electrical charge or molecules with the same charge as the ion-exchange support) are removed by washing, and bound biomolecules are recovered by elution with a buffer of either higher ionic strength or altered pH.

Proteins are complex ampholytes whose charges depend on the proportions of the amino acid residues in their structure as well as the pH of buffer. The isoelectric point (pI) of a native protein depends on the structural proportion of ionizable amino acids, their environment in 3D structure. Positive charges are typically found when the pH of the protein solution is below 8, due to the N-terminal amine and basic residues (arginines and lysines). Similarly, negative protein charges typically exist above pH   6 and are due to the C-terminal carboxyl group and acidic (e.g., aspartate and glutamate) residues. The charged groups are usually on the surface of proteins, except in case of metalloproteins, where the metal ion (usually inside the molecule) is usually coordinated by ligands (amino acid residues of the protein) [11].

A stoichiometric model describes the relationship between the charged groups in a protein and the stationary phase. The number of charged groups of the protein binds to the same number of oppositely charged groups of an ionic exchanger, and counterions are released both from the protein and the ion exchanger (see Fig. 5.3). Protein retention on an ionic surface depends on the protein charge, surface charge, and the charge characteristics of the surrounding medium. To describe this phenomenon, Kopaciewicz et al. [12,13] developed a nonmechanistic model that shows a positive correlation between protein retention and the number of charges associated with the adsorption–desorption process. Attention must be paid to the selection of buffer suitable for IEX. Buffer counterions can bound to IEX column and decrease its capacity and even change selectivity.

Generally, anion-exchange chromatography (AEX) is used at pH values above the isoelectric point of the protein of interest, while cation-exchange chromatography (CEX) is performed below the isoelectric point. At low pH or even at very high ionic strength, proteins may adsorb very strongly to an ion exchanger. This probably occurs due to an increase in the number of hydrogen bonds [11].

The interaction between a protein and an ion exchanger depends not only on the net charge and the ionic strength but also on the surface charge distribution and conformation of the protein. Some structural changes can affect the separation by IEX. Urea is widely employed to facilitate protein separations in ion-exchange chromatography at various scales. Hou et al. [14] indicated that the retention times correlate well with structural changes and they are more sensitive to the change of the tertiary structure.

The properties of the ion exchanger also influence the protein separation. Depending on the functional group, ion exchangers are classified as weak or strong. Use of strong ion exchangers, such as sulfonate (CEX) and quaternary ammonium (AEX), with pKa values outside the pH range for work with proteins (i.e., pH   4–10) result in the charge of the ion exchanger remaining the same despite changes in mobile-phase pH. These ion exchangers are applicable in case of weakly ionizable proteins. The benefits of the use of weak ion exchangers are related to a reduced tendency for sample denaturation, less ability to bind impurities, and enhanced resolution.

In general, two methods are applicable in the elution strategy in IEX: changing the pH of the eluting buffer and increasing the ionic strength by addition of salts, mostly NaCl.

The most common active groups related to the ion-exchange chromatographic matrices are carboxyl (–COOH), sulfonyl (–SO3H), secondary or tertiary amines (–NH2, –NRH), and a quaternary amino group (–N+R2H). The ion exchangers are classified as weak-cation, strong-cation, weak-anion, and strong-anion exchangers, respectively.

Lendero et al. [15,16] studied a universal nondestructive, noncontaminating method for the characterization of ion-exchange chromatographic columns. This method is based on making a step change in ionic strength of buffer solutions with the same pH in the ion-exchange columns and can be used for identification and determination of the type of ion-exchange groups on all sorts of ion exchangers (see Fig. 5.4). This investigation resulted in the observations that after the step change from Tris–HCl buffer to Tris–HCl buffer with sodium chloride, (1) the effluent pH for strong-anion-exchange columns remains nearly the same or rises by <   0.5   pH units in the shape of sharp and relatively short peak; (2) for weak-anion-exchange columns, it raises >   1   pH unit and this lasts several column volumes (CV); (3) for cation-exchange columns, it drops by >   1   pH unit.

Fig. 5.4. A scheme to distinguish among different active groups on the most common ion exchangers. To distinguish among different ion exchange groups on the convection interaction media supports (CIM), Tris–HCl buffer pH   7.4 was pumped through the column. The change in pH values of the solution (Tris–HCl buffer with NaCl) and at the column outlet were measured. The pH profiles versus elution volume normalized to the column volume were compared in three parts of the profile: (A) at low ionic-strength buffer (the effluent pH for the weak anion–exchanger DEAE was lower than for the other ion exchangers), (B) at high ionic strength buffer (the pH of the cation exchangers reduced, whereas the pH of the strong anion exchanger remained the same), and (C) the two cation exchangers were classified by the duration of the pH drop (for the weak cation exchanger, the pH drop lasted longer than for the strong cation exchanger).

 Printed from the Lendero N, Vidič J, Brne P, Frankovič V, Štrancar A, Podgornik A. Characterization of ion exchange stationary phases via pH transition profiles. J Chromatogr A 2008;1185(1):59–70 with permission.

In practice, the strategy based on the ionic strength by the addition of NaCl is the method of choice. As a rule, more weakly charged proteins are eluted at lower salt concentrations, whereas the more strongly charged proteins are eluted at higher salt concentrations. Stepwise elution is often used for the recovery of a concentrated protein especially in preparative chromatography. In this case, the optimization of gradient conditions is needed [17–19]. Optimized step gradient can be exploited in separation of proteoforms that exhibit different charge density at the surface [20] as a consequence of different amount of charged groups attached to oligosaccharide component [21,22].

One of the most used separation techniques in protein purification is IEX, for an advantage of this technique is that the elution normally takes place under mild conditions, and the protein can maintain its native conformation during the chromatographic process. Limited selectivity is the major disadvantage of this method [23].

To establish the structural and functional relationships in the characterization of all human proteins, of greatest importance are proteins of the blood. Blood plasma contains an unusually small group of high-abundance proteins, namely, serum albumin, immunoglobulins, transferrin, and α-2-macroglobulin. The amount of these proteins is about 85% of the total serum protein, and they often interfere with the identification of proteins of lower abundance. In proteomics, characterization of the blood proteome requires extensive fractionation before mass spectrometry analyses, and the removal of high-abundance proteins and subsequent enrichment of low-abundance proteins is a crucial step. For this purpose, IEX can be combined with other chromatographic methods, mostly with SEC and chromatography on hydrophobic resins [24,25].

As shown in Fig. 5.5, CEX and AEX, used after SEC and before hydrophobic-interaction chromatography (HIC), were applied in the separation of human plasma proteins. After the initial fractionation steps using ammonium sulfate precipitation and SEC, in the next steps, cation- and anion-exchange chromatography resulted in the highest number of identified proteins in the human plasma proteome [26].

Fig. 5.5. (A) Cation-exchange chromatography trace (UV 280   nm, black line) of the interim Analyte Library fraction of 65%-G3 (65% ammonium sulfate saturation, third pooled gel-filtration fraction). Conditions: HiTrap SP HP column with 0–0.5   M KCl (in 50   mM phosphate buffer, pH   5.5) gradient elution, flow rate: 1   ml/min, 1-ml fractions. (B) Anion-exchange chromatography trace (UV 280   nm, dark line) of the cation-exchange chromatography flow-through of fraction 75%-G4 (75% ammonium sulfate saturation, fourth pooled gel filtration fraction). Conditions: HiTrap Q HP column, 0–0.5   M KCl (in 20   mM Tris–HCl, pH   8.5) gradient elution; flow rate: 1   ml/min, 1-ml fractions.

 Reprinted from Kovács A, Sperling E, Lázár J, Balogh A, Kádas J, Szekrényes A, et al. Fractionation of the human plasma proteome for monoclonal antibody proteomics-based biomarker discovery. Electrophoresis 2011;32(15):1916–1925 with permission.

In proteomics, multidimensional chromatographic separation and analysis of the proteins are performed at the peptide level, after proteolytic digestion of the entire proteins extracted from tissue samples (the bottom-up approach) [27]. Strong cation-exchange chromatography (SCEX) is one of the frequently used liquid-chromatography strategies, where it has been shown that peptides are eluted according to their charge in a defined process. Compared with the classical strong cation exchange followed by ion-pair reversed-phase liquid chromatography (SCX   ×   IP–RPLC) approach, the reversed-phase followed by ion-pair reversed-phase liquid chromatography (RP   ×   IP–RPLC) showed a more homogenous distribution of eluted peptides at high pH [28,29].

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Blood Proteomics

M.A. Karpova , ... A.I. Archakov , in Journal of Proteomics, 2010

Lopez et al. used mass-spectral profiling of albumin-bound peptides for ovarian cancer biomarker discovery. They used Cibachron blue dye affinity chromatography-based technology on ZipPlates to bind high-abundant proteins. MALDI mass spectra were obtained. Both fragments of major proteins (transthyretin, complement, fibrinogen) and less abundant proteins potentially involved in cancerogenesis (kaseinkinase 2, transgelin) were present among discriminative peaks [51]. As a result, Lopez et al. discovered biomarker panels that could distinguish the first stage of ovarian cancer from unaffected patients with no evidence of ovarian cancer, with a sensitivity of >   93% and specificity of 97%.

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Intelligent thermoresponsive polymeric stationary phases for aqueous chromatography of biological compounds

Akihiko Kikuchi , Teruo Okano , in Progress in Polymer Science, 2002

PIPAAm's soluble/insoluble changes have been recently utilized to control affinity of target molecule by modulating the surrounding temperature [69]. In this system, adsorbed biomolecules could be completely recovered by lowering temperature through 'kicking-out' effect of samples with expanding PIPAAm chains (Fig. 16) . To do this, Cibacron Blue is immobilized onto amino functionalized polymethacrylate bead matrixes with spacers having different spacer sizes. Cibacron Blue is known to have higher affinity to serum albumin molecules and used in dye-affinity chromatography [70,71]. The matrix surfaces are co-immobilized with end-carboxyl PIPAAm. End-carboxyl PIPAAm was synthesized through radical telomerization using MPA as chain transfer agents in N,N-dimethylformamide (DMF) [46]. The PIPAAm chain lengths are controlled by the molar ratio of thiol compounds to IPAAm monomer. In our study, the molecular weight of PIPAAm was 1900 with molecular weight distribution index of 3.8 as determined by terminal carboxyl quantification by titration and GPC measurement. Polymethacrylate beads with epoxy side chains are aminated with 1,6-hexamethylenediamine for further conjugation of spacer molecules and active esterified PIPAAm molecules. Spacers used were 1,3-butadiene diepoxide and ethylene glycol diglycidylether. Those spacer molecules were used to change the distance between bead surfaces and affinity molecule, Cibacron Blue. Assuming that the surface-grafted PIPAAm is fully expanded below the LCST, its length is comparable or slightly longer than the Cibacron Blue bound with 1,3-butadiene diepoxide. However, in case of ethyleneglycol diglycidylether as the spacer, the length of Cibacron Blue is much longer than the fully expanded PIPAAm molecules. Thus, by changing temperature, the number of affinity molecules on the bead surfaces could be regulated. In fact, no albumin adsorption onto PIPAAm-grafted surfaces was evident, however, temperature dependent adsorption was obvious for Cibacron Blue co-immobilized beads surfaces. At higher temperature than PIPAAm's LCST, albumin adsorption is large, and the amount decreased with lowering temperature below the LCST. Adsorbed albumin at higher temperature was easily desorbed with low temperature treatment, where PIPAAm molecules hydrate and expand to outward. Expansion of PIPAAm molecules induced albumin conjugated to Cibacron Blue to be pushed out. Almost all albumin molecules on the Cibacron Blue immobilized with 1,3-butadiene diepoxide spacers were desorbed with only decreasing temperature. No modification to eluent pH, or ionic strength is applied. When ethylene glycol diglycidylether was used as spacer molecule for Cibacron Blue immobilization, the limited number of albumin adsorbed on the bead surfaces can be desorbed with lowering temperature. This is probably due to the insufficient expansion of PIPAAm molecules at lower temperature since the expanded chain length of hydrated PIPAAm is calculated to be shorter than Cibacron Blue with ethylene glycol diglycidylether spacers. Thus, the size of spacer molecules and PIPAAm chain length are both dominant factors for controlled albumin adsorption/desorption behavior.

Fig. 16. Schematic representation of the concept for selective adsorption/desorption control with thermoresponsive PIPAAm co-grafted with affinity dye, Cibacron Blue.

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