P r o P a c ® I M A C - 1 0 C o l u m nS o l u t i o n s f o r P r o t e i n a n d P e p t i d e A n a l y s i s

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HPLC column for high-resolutionimmobilized metal affinity chromatog-raphy (IMAC) separations of metal-binding proteins:• State-of-the-art technology for

tailored specificity• Protein capture, separation, and

elution from one column in asingle run

• Retention control by imidazole orpH gradient

• High purity separations of metal-binding proteins

• Wide range of metal-specificapplications

HPLC Separation of ProteinsUsing IMAC

Protein structure varies widely,presenting challenges in proteinanalysis and characterization. As aresult of this complexity, a variety oftools have been developed to isolate asingle protein, or a small group ofproteins from a larger mix, based on aunique characteristic of that singlemolecule or sub-group. IMAC is onesuch tool.

IMAC is a powerful purificationtechnique that separates proteins basedon their affinity for metals. Proteinswith an affinity for the metalimmobilized on the resin surface areretained, while other proteins are eluteduninhibited. The Dionex ProPac

IMAC-10 column, the latestadvancement in IMAC technology, is atrue high-pressure, high-resolutioncolumn capable of not only separatingcertain metal-binding proteins fromnon-metal binding proteins, but alsoproviding resolution of retained proteinsfrom one another, in many cases.

The ProPac IMAC-10 column can beapplied to a broad range of applications,including the separation of His-taggedprotein aggregation variants, His-taggedprotein purification, polishing samples forcrystallization experiments, on-columnrefolding, phosphopeptide analysis,intact protein separations, separation ofmonoclonal antibodies, and prionpeptide analysis.

ProPac IMAC-10 Resin Surface Showing Single Nanoparticle-Protein Interactions

wesrawlinsThermo

Characteristic ValueSubstrate Polystyrene divinylbenzene

Particle size 10 µm

Substrate x-linking 55%

Porosity Non-porous

Functional Ligand Iminodiacetate

Chelating metals Ni, Fe, Cu, others

Metal Ion Capacity ~40 µmol Cu/g resin

Protein Binding Capacity 1–5 mg lysozyme/g resin

pH stability 2–12

Backpressure limit ≤3,000 psi

Temperature limit ≤ 60 °C

Storage solution 20 mM MES, 141.8 mM NaC1,

1 mM EDTA pH = 6.10 + 0.1% NaN3

State-of-the-Art Technology forTailored Specificity

Typical IMAC stationary phasesare based on soft-gel matrices, such asagarose or cross-linked dextran. Whilecommercially available IMAC resinsare effective for low-pressure appli-cations, they exhibit low mechanicalstrength and are not amenable to evenHPLC applications. More recently,rigid polymer matrices have beendeveloped that are able to withstandhigher backpressures, typical of FPLC.However, the resolution possible withthese resins is such that often thecaptured fractions will require furtherpurification. The Dionex ProPacIMAC-10 column is a true HPLCcolumn capable of performing a largenumber of injections without losingcapacity. When the capacity does startto decrease, it can be recovered simplyby recharging the column with metal.

To produce this HPLC IMAC phaseDionex scientists coated 10-µm,nonporous, polymeric beads with ahydrophilic layer, to eliminate secondaryhydrophobic interactions with theproteins. Next, using state-of-the-arttechnology, isolated, poly(IDA) graftswere engineered to the surface of thesenonporous, polymeric beads. Thepoly(IDA) grafts are converted to metal-containing nanoparticles when thecolumn is charged with metal, asillustrated schematically in Figure 1. It isthese nanoparticles that act as the IMACinteraction sites for individual proteins.

The transmission electronmicroscopy image of the IMACbead is shown in Figure 2. Theimage reveals the bead interior, thehydrophilic layer, and the coppernanoparticles. These nanoparticlesare covalently bound to theexterior of the hydrophilic layer.Since the average diameter ofproteins is similar to the averagediameter of the surface boundnano-particles, a single proteininteracts with a single nano-particle. This interaction optimizescolumn performance, on-columnrefolding applications, and site-specific biotinylation experiments.

Figure 1. Formation of metal-containing nanoparticles upon charging of the ProPacIMAC-10 column with metal.

Figure 2. Nano-engineered stationaryphase using state-of-the-art technology.

TABLE 1 . PROPAC IMAC-10 RES IN CHARACTER IST ICSThe Dionex ProPac IMAC-10

column is stable at pH 2–12 andcompatible with most reagentscommonly used in protein purification,including denaturants, non-ionicdetergents, and reducing agents. It canalso be used under native or denaturingconditions using HPLC. The charac-teristics of the ProPac IMAC-10 resinare listed in Table 1.

Capture, Separate, andElute in a Single Run

IMAC is typically carried outusing a capture/release method forprotein and peptide enrichment. Often,the fractions collected contain impuri-ties that require further purification byreversed-phase HPLC, ion-exchange, orsize exclusion. Figure 4 shows anexample of a His-tagged protein thathad previously been purified by thisapproach. The ProPac IMAC-10analytical column allows researchers torun gradient separations in the IMACmode, resulting in highly efficientresolution of proteins and highly purefractions collected.

Retention Control by Imidazoleor pH Gradient

The protein adsorption step inIMAC is performed between pH 6 andpH 9; nonspecific adsorption is reducedby adding high concentrations of salt tothe binding buffer. The elution step canbe performed in one of three ways:using a pH gradient; introduction of acompetitor ligand; or by stripping themetal from the column. The DionexProPac IMAC-10 column is stable atpH 2–12 and thus compatible with a pHgradient. However, if a decreasing pHgradient is chosen, protein sensitivity tolow pH must be considered.

The recommended method foreluting bound protein from a DionexProPac IMAC-10 column is a linearimidazole gradient, as illustrated inFigure 3. Using a linear gradient ratherthan a step gradient allows for not onlythe elution of bound proteins, but alsothe separation of different boundfractions at different imidazoleconcentrations.

Figure 3. Retention control by imidazole or pH gradient.

High-Resolution Separation of ProteinsDionex columns are all

characterized by their ability to deliverhigh resolution separations, and theProPac IMAC-10 is no exception. Theuse of small, non-porous resin particlesresults in narrow, high efficiency peaks,with minimal band broadening due todispersion. The hydrophilic layersurrounding the resin particleeliminates peak broadening due to non-specific hydrophobic interactions.

The capacity of the column ismaintained by grafting the polymerchains that carry the functional ligandonto the resin. This design maximizesthe number of sterically accessibleligands for binding to the proteins,thereby ensuring that the proteinsremain tightly bound during theseparation step. Together, these designfeatures result in high-efficiency peaks,enabling the ProPac IMAC-10 toresolve proteins that cannot be resolvedon other IMAC resins.

Using an IMAC Phase1. Charge with copper2. Rinse off excess copper3. Load protein4. Elute protein with additive gradient or pH gradient

M

Gradientelution

Surface-exposedhistidine

Immobilized metal

Chelatingfunctionalitycovalently bound

200 30 40 60

AU

0.00

0.05

0.10

molar m

ass (g/mol)

1.0 x 105

1.0 x 10

UV trace(280 nm)

LS trace

60kD

110kD

180kD

280kD

0.15

Minutes

1 2 3 4 5 6 7

S 1 2 3 4 5 6 7 8 9

Column: ProPac IMAC-10 (4 mm x 250 mm)E1: 20 mM HEPES + 0.5 M NaC1, pH = 7.5E2: A + 500 mM imidazole, pH = 7.5Gradient: t (min) %A %B 0 95 5 80 0 curve 7 100 100 0 100 Inject Vol: 100 µLSample: His-Tagged ProteinFlow Rate: 0.5 mL/minWavelength: 280 nm

Peak #:sequence

1 2 3 KKRPKP 4 KKRP 5 KKRPKPWGQ

PHGGGWGQPHGGGWGQ PHGGGWGQPHGGGWGQ (G1, G4 methylated)

PHGGGWGQ PHGGGWGQ (G1 methylated)

PHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQ PHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQWGAPHGGGWGAPHGGGWGAPHGGGWGAPHGGGWGA6

7

Column: ProPac IMAC-10 (4 mm x 250 mm)E1: 20 mM HEPES + 0.5 M NaC1, pH = 7.5E2: E1 + 500 mM imidazole, pH = 7.5Gradient: t (min) %B 0 95 10 0 curve 7 30 0 Inject Vol: 15 µLSample: Prion-derived peptidesFlow Rate: 0.5 mL/minWavelength: 280 nm

24 26 28 30-0.007

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.00

23

1

5

4

6

7

1 repeat2 repeats

4 repeats

5i

6i4i

3i

2 4 60 8 10 12 14 16 18 20 22

Minutes

AU

Figure 5. Separation of prion related peptides.

Wide Range of Metal-SpecificApplications

His-tagged Protein AggregationVariants

His-tagged proteins are usuallypurified with an IMAC cartridge byapplying a capture/release protocol.The purity of the released fraction istypically assessed by SDS-Page. SDS-Page often reveals the presence ofprotein impurities from the host system(i.e., Escherichia coli) after IMACcartridge purification (Figure 4). Theamount of impurity can be reduced byoptimization of rinse conditions;however, impurities with affinitysimilar to, or greater than, His-taggedprotein can not be excluded by thisapproach. Using the Dionex ProPacIMAC-10 column and a linearimidazole gradient, resolution betweendifferent prion peptides can be achievedas shown in Figure 4. Monitoring theseparation with a UV detector allowscollection of the fraction containingHis-tagged protein, while the impuritiesare passed on to waste.

Aggregation is a problem for someHis-tagged proteins. The chromatogramin Figure 4 shows an imidazole gradientseparation of a 55-kD His-taggedprotein that is known to polymerize, andwas previously purified by IMACcartridge capture/release. The separationwas carried out using non-denaturingconditions that mimic proteinpolymerization conditions. The multiplepeaks shown in the trace were verifiedas aggregation variant peaks by on-linelight-scattering detection. Prion Peptides

The ability to capture and separatepeptides with affinity for immobilizedcopper in one run and on one column isadvantageous for proteomicsapplications. The proteomics approachinvolves identification of large numbersof proteins in very complex mixtures.Typically, the mixtures are simplified byclass separation. IMAC can be used tocapture the copper binding peptide class.

Figure 5 shows a separation of sixsynthetically prepared copper bindingpeptides related to prion protein. Thepeptides contain octapeptide repeats

where each repeat has been found tobind one copper atom. Using theProPac IMAC-10 column, prion-relatedpeptides differing in the number ofoctapeptide repeat units(PHGGGWGQ) (peak 1),(PHGGGWGQ)2 (peak 3), and(PHGGGWGQ)4 (peak 5) wereseparated. The column is also able toseparate several prion-related peptidemixtures where the peptides containedthe same number of copper bindingsites but differed in the presence of ahydrophilic tail (peak 5 and peak 6) orQ_A mutation (peak 6 and peak 7).

Figure 4. Separation of His-tagged protein aggregation variants.

20418

20420

Column: ProPac IMAC-10 (4 mm x 250 mm)E1: MES + NaCI, pH = 6.0E2: E1 + imidazole, pH = 6.0Gradient: t (min) %A %B 0 96 4 60 0 100 80 0 100Inject Vol: 15 µLSample: 1 — 1.0 mg/mL MAb PDLFlow Rate: 0.5 mL/minWavelength: 280 nm

50454035302520151050Minutes

-0.001

0.005

0.010

0.000

1

AU

Figure 6. Monoclonal antibody.

Monoclonal AntibodiesMonoclonal antibodies (MAbs) are

used in a wide variety of applications,each of which has its own set of purityrequirements. Figure 6 shows thechromatographic trace of an very pureMAb sample. The ability to isolateindividual proteins on individualnanoparticles is expected to result inhigher purity fractions from site-directed biotinylation experiments

Intact Protein SeparationsAnalytical IMAC is capable of

separating proteins based on the extentof surface exposed histidine residues.The chromatogram in Figure 7 showsresolution of three standard proteinsseparated on the ProPac IMAC-10column using an imidazole gradient.

-0.002

0.010AU

0.020

0.000

1

2

3

Column: ProPac IMAC-10 (4 mm x 250 mm)E1: 20 mM HEPES + 0.5 M NaC1, pH = 7.5E2: E1 + 100 mM imidazole, pH = 7.5Gradient: t (min) %B 0 96 15 0 curve 7 40 0 Inject Vol: 15 µLSample: 1 – 0.25 mg/mL ribonuclease A 2 – 1.00 mg/mL myoglobin 3 – 1.50 mg/mL carbonic anhydrase Flow Rate: 0.5 mL/minWavelength: 280 nm

0 5 10 15 20 25 30 35 40Minutes

Proteins with surface exposed histidines

ribonuclease A myoglobin

Figure 7. Separation of standard proteins within the same class.

20483

20416

Phosphopeptide Capture and ReleaseDetection of phosphorylation in

proteins is necessary to theunderstanding of their biologicalfunctions, but the abundances areusually low, making this a challenginganalytical problem. IMAC in the ferricform has been used on enzymaticdigests of phosphorproteins toselectively fractionate phosphorylatedfrom nonphosphorylated peptides.Tryptic digests of beta-casein wereevaluated to determine nonspecificbinding of nonphosphorylated peptidesand recovery of phosphopeptides, asshown in Figure 8. Fractions wereanalyzed by reversed-phase HPLC. Thechromatograms in Figure 8 show theun-fractionated digest, released fraction(phosphopeptide enriched), and flow-through fraction (nonphosphorylatedpeptides). The captured peptides wereverified as phosphopeptides bydephosphorylating and monitoring runtime shifts.

3 5 10 162.60

IMAC RP-HPLC

Minutes

–200

1,800

–40

250

mAU

Unretained fraction, beta-casein digest

Retained fraction, beta-casein digest

Figure 8. Phosphopeptide analysis.

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PROPAC IMAC-10 COLUMNS ORDER ING INFORMATION

In the U.S., call 1-800-346-6390, order on-line at http://dstore.dionex.com,or contact the Dionex regional office nearest you. Outside the U.S., order throughyour local Dionex office or distributor. Refer to the following part numbers.

Product Description Part Number

ProPac IMAC-10 Column (1 × 50 mm) ..................................................... 063617

ProPac IMAC-10 Column (2 × 50 mm) ..................................................... 063272

ProPac IMAC-10 Column (4 × 50 mm) ..................................................... 063276

ProPac IMAC-10 Column (9 × 50 mm) ..................................................... 063615

ProPac IMAC-10 Column (4 × 250 mm) ................................................... 063278

ProPac IMAC-10 Column (9 × 250 mm) ................................................... 063280

ProPac IMAC-10 Column (22 × 250 mm) ................................................. 063282

Guaranteed performanceThe unique pellicular resin of the

ProPac IMAC-10 columns offersexceptional selectivity and stabilityover the entire pH range. Its highlycrosslinked structure ensures longcolumn life and easy cleanup. Theentire manufacturing process (resinsynthesis, synthesis of the polymerchains, and packing and testing of thechromatographic columns) is carefullycontrolled to ensure that every DionexProPac IMAC-10 column deliversreproducible performance. ProPacIMAC-10 columns are tested in thecopper-loaded mode with a standardprotein mix to ensure lot-to-lotreproducibility.

LPN 1731 10M 08/05© 2005 Dionex Corporation

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