-
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
colu
mns
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.
21563
-
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
Dionex Corporation Dionex Corporation Dionex U.S. Regional
Offices Dionex International Subsidiaries1228 Titan Way Salt Lake
City Technical Center Sunnyvale, CA (408) 737-8522 Australia (011)
61 2 9420 5233 Austria (01) 616 51 25 Belgium (32) 3-353 42 94
Canada (905) 844-9650 China (852) 2428 3282P.O. Box 3603 1515 West
2200 South, Suite A Westmont, IL (630) 789-3660 Denmark (45) 36 36
90 90 France (01) 39 30 01 10 Germany (06126) 991-0 Italy (06) 66
51 50 52 Japan (06) 6885-1213Sunnyvale, CA Salt Lake City, UT
Houston, TX (281) 847-5652 Korea 82 2 2653 2580 The Netherlands
(0161) 43 43 03 Switzerland (062) 205 99 66 United Kingdom (01276)
69172294088-3603 84119-1484 Atlanta, GA (770) 432-8100(408)
737-0700 (801) 972-9292 Marlton, NJ (856) 596-0600 * Designed,
developed, and manufactured under an NSAI registered ISO 9001
Quality System.