Use of Heparin HyperD® M Affinity Resin for Batch Mode Protein Purification From Plasma
Masilamani Selladurai, Saurabh Nagpal, Hongshan Li, and Lisa Bradbury; Pall Corporation, Bangalore, India and Woburn, MA, USA
MATERIALS AND METHODS (continued)
Heparin HyperD M affinity resin uses heparin as a mixed-mode affinity/ion exchange ligand for enrichment and
1. Approximately 500 µL of settled resin is washed with 3x 5 mL of 20 mM Tris-HCl, pH 7.4, in a 15 mL conical
separation of many important plasma proteins. Heparin is well-known to interact with coagulation factors, lipopro-
tube. Remove excess buffer by centrifugation at 800 x g for 4 minutes. An equal volume of fresh buffer is then
teins, growth factors, and growth hormones. Heparin binds to growth factors and antithrombin III (AT III) through
added to generate a 50% slurry.
specific affinity (three dimensional structure of binding sites). Heparin also has a high content of anionic sulfate
groups, which bind to positively charged proteins. As with ion exchange resins, salt and pH affect both binding
and elution of specific proteins. The combined properties of affinity and ion exchange allow purification and separation of proteins having a wide range of concentrations. In this study, binding and elution of AT III from 50 µL of
whole plasma is used as an example of single step purification. Adjusting salt concentration in the binding and
RESULTS (continued)
Figure 3
SDS-PAGE Analysis of Fractions From AT III Purification From Human Plasma Using Heparin HyperD Resin in an
AcroPrep 96-well Filter Plate with 300 mM NaCl Binding Conditions
)
Da
(k
W WP
M
2. Place an AcroPrep 96-well 350 µL filter plate on a vacuum manifold (PN 5017) with a 96-well collection plate
underneath to collect flow through.
3. 0.1 mL of resin slurry (50 µL resin) is quickly pipetted into wells on the filter plate.
4. Remove excess liquid by applying 10-20 cm vacuum (used for all the steps) for 5-10 seconds.
RESULTS (continued)
h
as
W
FT
1
l
Cl
Cl
aC
Na
Na
N
h
III
M
M
M
0
as
0
5
AT
2.
1.
W
1.
3
205
97
66
5. The resin is equilibrated with 2x 250 µL (500 µL) equilibration buffer for 5 minutes. Remove excess liquid
elution steps is critical for best results.
1-15 µL (150-200 ng) of sample loaded onto 12% SDSPAGE gels (BIORAD) run with glycine buffer. Lanes:
MW=molecular weight markers; WP=whole plasma; FT=flow
through; Wash 1=wash fraction 1; Wash 3=wash fraction 3;
1.0 M NaCl eluate, 1.5 M NaCl eluate, 2.0 M NaCl eluate,
and AT III=300 ng pure AT III. Coomassie Brilliant Blue G250 (GE Healthcare) stain for detection. Arrow mark shows
the protein band used for AT III identification using MALDI.
43
after incubation.
6. Dilute 50 µL of human plasma sample with 150 µL of binding buffer (1/4 dilution) and add to each resin filled
INTRODUCTION
well. Approximately 2 mg total protein is used for 50 µL of resin bed.
29
7. Thoroughly mix sample in a plate mixer for 20 minutes at room temperature to facilitate binding.
Traditional chromatography methods, such as size-exclusion, ion exchange, and reversed-phase chromatography,
are not highly efficient for purification of many important plasma proteins found at low concentration. Affinity chromatography is a preferred method for specific capture and purification of these classes of proteins.
Heparin is a negatively-charged mucopolysaccharide consisting of a repeating dimer of hexuronic acid and
D-glucosamine with a molecular weight range of 5,000-30,000 (Figure 1). Heparin has two modes of interaction
with proteins:
1. As an affinity ligand it will bind specifically with proteins involved with blood clotting and growth factors.
2. As a cation exchanger due to its high content of anionic sulfate groups. For example, heparin will interact with
8. Flow through fractions containing unbound proteins are collected.
20
9. Wash resin with 4x 250 µL (20x resin volume) equilibration buffer.
10. Retained proteins are eluted with a binding buffer with progressively increasing salt concentration. Two times
150 µL of each elution buffer is used (= 3 resin volumes) to ensure good elution efficiency and to minimize
sample dilution. The protein is only found in the first elution fraction, thus elution appears to be quite efficient.
Figure 2
Flow Chart for Heparin HyperD M Resin for AT III Purification
14
Due to the strong charged nature of heparin, salt dramatically influences binding and elution behavior. Salt inhibits
15
10
5
ion exchange interactions with heparin while having minimal impact on affinity interactions. When the plasma
sample contains only 100 mM NaCl during the binding step, there is a significant increase in the number of different
proteins that bind, as seen in the eluate fractions in Figure 4 compared to eluate fractions in Figure 3. Albumin
lower salt elution buffer (e.g., 0.5 M), but in this case it is easier to prevent binding of impurities than to selectively
Wash resin with equilibration buffer
remove them from the resin after binding. Hence, NaCl is increased from 100 to 300 mM NaCl during the binding
0
100
50
150
200
Probability-based Mowse Score
Probability-based Mowse score and AT III coverage map (matching peptides in bold red font). MASCOT Peptide Mass Fingerprint used
for protein identification.
step. This data illustrates the potential for controlling binding and elution with salt.
Hexuronic Acid
COO–
Figure 4
SDS-PAGE Analysis of Fractions From AT III Purification From Human Plasma Using Heparin HyperD Resin With
Lower Salt (100 mM NaCl) Binding Conditions
Re-suspend resin as 50% slurry
and load resin into plates
D-glucosamine
Acid
O
20
does not show significant heparin binding. This large group of proteins could be separated from the AT III with a
nucleic acid binding proteins as its structure mimics the polyanionic backbone of nucleic acids.
Figure 1
Heparin Chemical Structure1
Figure 6
MASCOT Peptide Mass Fingerprint Results
Number of Hits
ABSTRACT
O
D-glucosamine
)
Da
(k
W WP
M
OH
Add plasma
H2OCR1
OH
R2
L-iduronic Acid
O
COO–
Collect wash fractions
OH
HNR2
R1 = –H or –SO3
OR1
plasma protein. Excellent purity is achieved using Heparin HyperD resin in 96-well filter plates and 0.5 mL
Nanosep® centrifugal devices (data not shown). AT III is retained at pH 7.4 with 300 mM salt. The bound protein
205
is eluted using 1.0-2.0 M salt. Since Heparin interacts with AT III as an affinity ligand, the binding of the protein
97
with 300 mM NaCl is very specific. The data demonstrates that Heparin HyperD resin can be used for single step
purification in batch mode of AT III from human plasma, a very complex protein mixture. The current method was
66
Wash with equilibration buffer
SDS-PAGE
and
MALDI analyses
Elute with high salt buffers
R2 = –SO3 or –COCH3
Heparin HyperD M resin was used in a batch mode 96-well format for the affinity purification of AT III. AT III has a
reported concentration of approximately 120 µg/mL in human plasma, which represents less than 0.25% of total
Cl
Cl
a
a
N
N
h
M
M
0
as
5
2.
W
1.
Collect FT fraction
OH
O
O
O
Incubate for
20 minutes
FT
DISCUSSION
1-15 µL (150-200 ng) of sample loaded onto 12% SDS-PAGE gels (BIORAD)
run with glycine buffer. Lanes: MW=molecular weight markers; WP=whole
plasma; FT=flow through; Wash=wash fraction; 1.5 M NaCl eluate; and
2.0 M NaCl eluate. Coomassie Brilliant Blue G-250 (GE Healthcare) stain for
detection.
43
29
optimized with low sample and resin volumes, although it can readily be scaled up. This study offers a simple and
efficient protocol for enrichment and purification of heparin binding proteins.
CONCLUSIONS
Collect eluate fractions
Due to heparin’s unique structure, individual proteins often bind by a combination of affinity and ion exchange,
The batch mode protein purification protocol using Heparin HyperD resin was developed:
20
and small differences between bound proteins can result in good separations. A key advantage of this chromatography
is that plasma proteins which bind to heparin can be conveniently enriched in a single step.
Heparin HyperD M affinity resin is a composite structure comprised of a porous rigid mineral bead containing
To achieve effective enrichment and purification of low concentration proteins such as AT III from human plasma.
Protein Digestion, MALDI Analysis, and Database Search
As a starting point for the enrichment and purification of other heparin binding proteins, such as
14
coagulation factors, growth hormones, growth factors, and lipoproteins, with minimal optimization of salt and
heparin bound in hydrogel filled pores. The high surface area in the pore structure of the beads offers high capacity
Putative AT III protein bands from SDS-PAGE eluate fractions were excised and used for in-gel trypsin digestion
for heparin binding proteins. A batch mode method simplifies the overall procedure because it does not use fluid
using manufacturer’s instructions (Sigma). MALDI analysis was performed using the ABI Voyager DE Pro MALDI-
Identification of AT III Using Peptide Mapping and Mass Spectrometry
pH conditions
flow, but instead relies on incubations in a container.
TOF instrument. 1 µL of digested protein was spotted onto a gold MALDI plate. CHCA in 50% acetonitrile + 0.1%
To confirm the identity of AT III in the eluate fraction, the protein band from the three eluate fractions (1.0, 1.5, and
As a sufficiently flexible platform to accommodate a range of sample volumes and protein loads.
TFA was used for detection. MASCOT Peptide Mass Fingerprint was used for the protein identification.
2.0 M NaCl) were excised from the gel and subjected to trypsin digestion. The resultant peptides were analyzed
These data demonstrate the effective purification and concentration of AT III from human plasma. The current pro-
using MALDI-TOF (1.0 M NaCl eluate, Figure 5). Forty-six peptide m/z values were used for a peptide map search,
tocol can also be used for fractionation of plasma or other proteins, which can be achieved by altering the salt
which identified the band as human AT III, with a probability score of 209 (> 78 is significant) and 44% coverage
concentration and/or pH of the binding buffer.
The objective of the current study is to utilize the benefits of Heparin HyperD M resin for the one-step purification
of AT III protein from small volumes of plasma samples. A simple protocol using Heparin HyperD M resin in 96well plates for the purification of AT III is presented.
RESULTS
of the complete amino acid sequence (Figure 6).
Analysis of chromatographic fractions indicates that AT III is retained at physiological pH with minimum salt,
although 300 mM NaCl is used to increase specificity. The bound protein is eluted with high salt (1.0-2.0 M). This
results in a single band on SDS-PAGE which is identified as AT III using MALDI-TOF analysis and Peptide Mass
Fingerprinting.
Heparin HyperD M Resin Effectively Purifies AT III From Human Plasma
With a Single Chromatography Step
REFERENCE
Figure 5
MALDI-TOF Analysis of In-Gel Trypsin Digest of 1 M NaCl Eluate Sample
100
8 4 2 .4
1. Gatti, G., Casu, B. et al. (1979). Studies on the conformation of Heparin by lH and
Heparin is known to have an affinity for the plasma protein AT III. This protein was chosen for the development of
Purification of AT III From Human Plasma
Human plasma – single donor, platelet poor plasma sample generated in Pall Corporation laboratories from
80
protein is expressed at relatively low levels. A method overview for AT III purification from human plasma using
Heparin HyperD M resin in an AcroPrep 350 µL 96-well filter is shown in Figure 2.
Analysis of the fractions (Figure 3) generated from this method indicates that AT III binds selectively to the resin
in the presence of 0.3 M NaCl and is eluted with high salt.
whole blood in K2EDTA. Plasma samples then aliquoted and stored frozen until use
AT III is selectively retained at pH 7.4 with 300 mM NaCl.
Heparin HyperD M resin (PN 20029-021)
The bound AT III is eluted with NaCl at 1.0 M and greater, although the highest purity is achieved with 1.0 and
1.5 M NaCl.
Buffers used for AT III purification:
The molecular weight of eluted AT III is similar to pure AT III (far right lane), and both are consistent with the re-
– Equilibration/binding buffer: 20 mM Tris-HCl pH 7.4 with 0.3 M NaCl
60
40
– Elution steps: 20 mM Tris-HCl pH 7.4 with 1.0, 1.5, and 2.0 M NaCl
Contact: Phone: 800.521.1520 (USA and Canada) • (+)800.PALL.LIFE (Outside USA and Canada) • www.pall.com • E-mail: LabCustomerSupport@pall.com
1 3 4 0 .5
1405. 6
8 5 0 .3
2 2 9 1 .0
1 528 .6
20
1 0 4 5 .5 1 1 2 6 .6 1 2 1 1 .5
1674 .7
8 0 0 .3
9 1 1 .4
ported molecular weights of 58 kDa (based cDNA) and 60 kDa (N-glycosylated form).
MALDI analysis of in-gel trypsin digest
of unique band from 1 M elution fraction
(Mr ~58KDa). Mass region (m/z) 799 to
2500 is shown.
123 2.6
9 1 7 .4
AcroPrep™ 96-well 350 µL filter plate (PN 5029)
C NMR spectroscopy.
Macromolecules 12 (5): 1001-1007.
a batch mode protein purification method as plasma is a challenging chromatography starting material and the
% Intensity
MATERIALS AND METHODS
13
1 0 2 2 .4
1 3 1 1 .4
1 8 7 4 .7
1 4 3 7 .6
2 1 9 9 .5
2 4 2 3 .0
1 9 9 7 .9
0
800
1140
1480
1820
2160
2500
Mass (m/z)
© 2009, Pall Corporation. Pall,
, AcroPrep, HyperD, and Nanosep are trademarks of Pall Corporation. ® indicates a trademark registered in the USA. 7/09, GN09.2922