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: [email protected]. At the time
of this writing, Patrick McCarthy was with Dionex Corp. but
is no longer with the company.