Analytical Separations of Proteins and Peptides Using Immobilized Metal Affinity Chromatography

Immobilized metal affinity chromatography (IMAC) is a well-established protein separation technique experiencing expanded use based on new, high-performance analytical phases. IMAC is commonly used in capture/release cartridges for enriching peptides and proteins of a certain class (i.e., surface exposed histidine [His] or phosphopeptides/phosphoproteins) or His-tagged recombinant protein purification. Due to the relatively low resolution of these cartridges, fractions collected from the cartridges can still be quite complex, and further purification is generally required. To address this limitation, a reusable, high-resolution IMAC column that can be used for high-performance (1-D and 2-D) LC separations has been developed.

The ProPac® IMAC-10 phase (Dionex Corp., Sunnyvale, CA) has been designed so that a single protein molecule can interact with a single nanoparticle on the surface of the bead. In this way, the captured protein is isolated from all other components in the matrix (i.e., proteins, peptides, DNA, etc.) and isolated from components bound to neighboring nanoparticles. Conceptually, this kind of design should be optimal for the separation of proteins or peptides within a class, on-column refolding, on-column protein–protein interaction studies, and on-column site-directed reactions (i.e., site-directed biotinylation). This paper demonstrates the resolving power of the ProPac IMAC-10 for several protein and peptide separations.

Experimental

Materials

Materials used were: sodium chloride (Fluka, Milwaukee, WI); imidazole (Fluka); urea (Sigma, St. Louis, MO); acetonitrile (Burdick & Jackson, Morris Township, NJ); deionized water, 18 MΩ•cm resistance, trifluoroacetic acid (TFA) (Pierce Chemical, Rockford, IL); tetrasodium pyrophosphate decahydrate (TSPP), Na4P2O7•10H2O (Sigma); formic acid, 98% (EM Science, Gibbstown, NJ); acetic acid glacial (J.T. Baker, Phillipsburg, NJ); ammonium hydroxide (Fisher Scientific, Pittsburgh, PA); iron(III) chloride hexahydrate, FeCl3•6H2O (Aldrich, Milwaukee, WI); β-casein (Sigma); and ProPac IMAC-10, 2 × 50 mm and 4 mm × 250 mm, and Acclaim 300 C18, reversed-phase, 4.6 × 150 mm (Dionex).

Instrumentation

Chromatography was performed on a Dionex instrument equipped with an AD20 detector, GP50 pump, AS50 autosampler, and Chromeleon® chromatography management software. The final example was performed using a Dionex dual-gradient HPLC system consisting of a P680 DGP6 pump, TCC-100 column oven, ASI-100T™ autosampler, UVD 340U detector, and Chromeleon chromatography management software.

Results and discussion

Figure 1 - Separation of the standard proteins with increasing number of surface-exposed histidine residues. The sample was eluted from the column at 0.5 mL/min with a gradient of 0–100 mM imidazole in 20 mM 2-morphinoethanesulfonic acid (MES), 500 mM NaCl, buffered to pH 6.0 with UV detection at 280 nm. Ribonuclease A, myoglobin, and carbonic anhydrase correspond to peaks 1, 2, and 3, respectively.

IMAC mechanism

The mechanism of protein separation in IMAC is coordination. In the case of IMAC-Cu, copper exposed on the resin surface provides coordination acceptor sites capable of binding surface-exposed His residues on the protein. Visual inspection of the crystal structures of ribonuclease A, myoglobin, and carbonic anhydrase show that they have approximately 4, 9, and 15 surface-exposed His residues, respectively. These proteins were separated by a gradient of increasing imidazole concentration (Figure 1).

His-tagged proteins (IMAC-Cu)

Figure 2 - Separation of IMAC cartridge purified His-tagged protein reveals residual impurities. The sample was eluted from the column at 0.5 mL/min with a gradient of 0–200 mM imidazole in 20 mM MES, 300 mM NaCl, 8 M urea, buffered to pH 5.5 with UV detection at 280 nm.

Recombinant protein purification is often achieved by capturing proteins engineered with His tags by IMAC cartridges. The protocol usually involves three steps: capturing with a binding buffer, rinsing off impurities, and eluting with a releasing buffer. Protein purity after cartridge purification is often insufficient for crystallization and important activity assays. The ProPac IMAC-10 column offers increased resolution for purification of His-tagged proteins. The column can be used for detecting and removing unwanted impurities (Figure 2).

Monoclonal antibodies (IMAC-Cu)

Figure 3 - Separation of intact monoclonal antibody. The sample was eluted from the column at 0.5 mL/min with a gradient of 0–100 mM imidazole in 20 mM MES, 500 mM NaCl, buffered to pH 6.0 with UV detection at 280.

As the number of variants on a single monoclonal antibody (Mab) increases (oxidation, deamidation, lysine truncation, sialylation, and aggregation), the sample heterogeneity increases dramatically. There are cation exchange, anion exchange, hydrophobic interaction (HIC), and size-exclusion chromatographic (SEC) methods to resolve MAb variants. However, the complexity of variants within a sample often requires the use of more than one chromatographic mode coupled with papain or trypsin digestion for variant separation and characterization. ProPac IMAC-10 in the copper mode is an effective complementary separation tool for this analysis. Figure 3a shows the separation of an intact monoclonal antibody. Figure 3b is a 10× magnification of Figure 3a, and shows that the ProPac IMAC-10 can resolve minor unknown impurities from the main MAb peak. After digestion with papain, ProPac IMAC-10 (Cu) can be used to separate Fab fragments from Fc fragments (data not shown).

Phosphopeptides (IMAC-Fe)

Figure 4 - Enrichment of phosphopeptides. The sample was bound to the ProPac IMAC column at 0.3 mL/min with 20 mmol formic acid and released from the column at 0.3 mL/min with 20 mmol formic acid titrated to pH 9.0 with ammonium hydroxide with UV detection at 214 nm. Fractions collected were reinjected on the Acclaim 300 C18 reversed-phase column using the following acetonitrile gradient—eluent A: 950 g water, 39.1 g acetonitrile, 0.44 g TSPP, 0.10 mL TFA; eluent B: 546 g acetonitrile, 300 g water, 0.11 mL TFA, gradient at 1.0 mL/min from 93% A to 31% A in 15 min with UV detection at 214 nm.

Beta-casein provides an excellent model protein to demonstrate the performance of the ProPac IMAC-10 column in the iron mode. When digested with trypsin, β-casein yields two phosphopeptides: a monophosphorylated (FQSpEEQQQTEDELQDK) and a tetraphosphorylated (RELEELNVPGEIVESpLSpSpSpEESITR). The ProPac IMAC-10 in the iron mode uses mobile phases that are directly compatible with a reversed-phase second-dimension separation (TN705). In this example, the complete 2-D separation was performed automatically using a Dionex dual-gradient pumpHPLC. The chromatographic results of automated IMAC enrichment and reversed-phase (RP)-HPLC analysis are shown in Figure 4. Figure 4a shows the unretained trace on the ProPac IMAC-10 and the reversed-phase separation of the unretained fraction (Figure 4b). Figure 4c shows the release of the peak that was retained on the ProPac IMAC-10 column and the reversed-phase separation of the retained fraction (Figure 4d). The monophosphopeptide and the tetraphosphopeptide elute at 5.8 and 9.2 min (Figure 4d), respectively.

Summary

The analytical ProPac IMAC-10 column gives protein chemists another powerful tool to separate complex biological mixtures. The columns are reusable, very robust in routine use, and deliver extremely high resolution for protein separations. With the metal most appropriate for the application, the user can charge the ProPac IMAC-10, which is shipped free from metal ions, and can be recharged for extended use. The column is stable at high pressure, allowing for increased flow rates and throughput. It supports multiple injections without recharging and is well suited for automated separations. The column is offered in several formats to serve both HPLC and fast protein liquid chromatography (FPLC) users.

Additional reading

Automated enrichment and analysis of phosphopeptides using immobilized metal affinity and RP chromatography with column switching. Tech. Note 705, LPN 1763. Dionex Corp., Jan 2006.

Separation of an intact monoclonal antibody and fractionation of monoclonal antibody papain digest fragments using immobilized metal affinity chromatography. Appl. Note 177, LPN 1840. Dionex Corp.

The authors are with Dionex Corp., 1228 Titan Way, Sunnyvale, CA 94088, U.S.A.; tel.: 408-481-4278; fax: 408-735-9413; e-mail: mark.tracy@dionex.com. At the time of this writing, Patrick McCarthy was with Dionex Corp. but is no longer with the company.

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