Specificity of High-Abundant Protein
Depletion Among Different Species with
the Agilent Multiple Affinity Removal
System
Application
Bio/Proteomics
Author
William Barrett
Agilent Technologies, Inc.
2850 Centerville Road
Wilmington, DE 19808-1610
USA
Abstract
The Agilent Multiple Affinity Removal System is quickly
becoming the technique of choice by leading scientists for
the rapid and simultaneous removal of multiple highabundant proteins from human serum or plasma. The
immunoaffinity-based removal of selected high-abundant
proteins with this system is accomplished with polyclonal
antibodies. The selectivity and specificity of these antibodies are optimized with a stringent affinity purification
procedure that uses highly purified human antigens. This
maximizes selectivity for the targeted proteins and minimizes nonspecific interactions and binding of nontargeted
proteins. This study examines the cross-reactivity of the
Multiple Affinity Removal System for human serum/
plasma with serum from several other species including:
Macaca Mulatta (Rhesus monkey), Canis Familiaris
(dog), Rattus Norvegicus (rat), Mus Musculus (mouse),
and Sus Scrofa (Swine). The results show that the optimized depletion of the six most abundant human proteins
is effective for Rhesus monkey serum, as determined by
gel electrophoresis. The results for dog serum showed
similar depletion of targeted high-abundant proteins, with
the exception of transferrin, as determined by gel electrophoresis. In the case of mouse, rat, and swine serum,
analysis of the flow-through fraction by gel electrophoresis shows that the targeted proteins are depleted, but still
present, indicative of incomplete removal. This study
demonstrates that the specificity and selectively of the
Multiple Affinity Removal System is uniquely optimized
for human proteins and is consistent with the previous
studies showing the highest possible selectivity for the
targeted proteins and low interaction with nontargeted
proteins.
Introduction
The Agilent Multiple Affinity Removal System is
being adopted by proteomic scientists who have a
need to remove multiple high-abundant proteins
from human serum or plasma simultaneously and
rapidly. The immunoaffinity column simultaneously removes six of the most-abundant proteins in
human serum using polyclonal antibodies. The
column removes greater than 98%–99% of albumin,
IgG, IgA, haptoglobin, transferrin, and antitrypsin
from human serum samples. As previously stated,
the immunoaffinity column is based upon polyclonal antibodies, which are purified via a stringent affinity purification method using highly
purified human antigens. It can be predicted that
such highly specific antibodies will only crossreact with proteins from closely related species
and would be less likely to interact with the same
proteins from less closely related species.
Recent studies have shown that the Agilent
Multiple Affinity Removal System works with cerebraspinal fluid (CSF) as well. However, no data has
shown the performance of the Agilent Multiple
Affinity Removal Column for various species. Many
other commercially available products do not
remove more than two high-abundant proteins,
and if cross-species reactivity is claimed, the
depletion efficiency tends to vary. Furthermore, if
cross reactivity occurs, one must investigate the
specificity of the depleted proteins. The stringent
purification process for the Agilent polyclonal antibodies eliminates the less specific antibodies that
may bind nontargeted proteins that are closely
related to the targeted proteins (for example,
human albumin is 72% identical to a lesser related
species such as rat; for reference, human albumin
is 92% identical to Rhesus monkey albumin). Scientists use many different animal models to investigate biomarkers, drug effects, and disease states
including monkey, dog, rat, mouse, and swine. This
note examines the specificity of the Agilent
Multiple Affinity Removal System for serum
derived from Rhesus monkey, canine, swine, rat,
and mouse sources relative to human serum.
Though proteins are depleted in the other samples,
the level of depletion is not complete and varies for
rat, mouse, and swine serum. This type of comparative study can provide an additional measure of
the specificity of the Multiple Affinity Removal
System for proteins from human serum/plasma.
Experimental
The Multiple Affinity Removal System for removing
albumin, transferrin, IgG, IgA, haptoglobin, and
antitrypsin from human serum is a product from
Agilent Technologies (Wilmington, DE). A
4.6 mm × 100-mm Multiple Affinity Removal
column was used to provide a larger serum capacity than the 4.6 mm × 50-mm column (Zhang et al.,
5989-0601EN). A mobile phase reagent kit
(Agilent Technologies) for the affinity column was
used for sample loading, washing, and column
regeneration (Buffer A) and for bound protein elution (Buffer B). Injections of diluted serum were
performed according to manufacturer protocols for
Figure 1.
2
Fraction Collector settings.
a 4.6 mm × 100-mm column. Fractions were automatically collected by peak detection into 1.5-mL
plastic tubes (Sarstedt, Numbrecht, Germany)
using an Agilent 1100 HPLC equipped with a thermostatted analytical scale fraction collector and
thermostatted autosampler, both set to 4 °C.
Depleted low-abundant proteins as well as highabundant bound proteins were collected and
stored at –20 °C until analysis.
Sample Analysis
Five species of serum were analyzed for highabundant protein removal efficiency according to
the standard LC protocol provided with the
Agilent Multiple Affinity Removal System. Briefly,
crude serum (human, Rhesus monkey, canine, rat,
swine, and mouse) were diluted five-fold with
Buffer A. The serum samples were filtered through
0.22-µm filters (Agilent part number 5185-5990) by
spinning at 16,000 × g at room temperature for
1.5 minutes. Diluted serum samples were placed in
an autosampler equipped to cool samples to 4 °C
and automated sample injection was set for 150 µL
per injection in 100% Buffer A at a flow rate of
0.5 mL/min for 10 minutes. The unbound lowabundant proteins were collected using an Agilent
thermostatted fraction collector set at 4 °C using
peak-based collection. The bound proteins were
eluted in 100% Buffer B at a flow rate of 1.0 mL/min
for 7 minutes. This step is followed by column
regeneration and equilibration in 100% Buffer A at
a flow rate of 1.0 mL/min for 11 minutes. Fraction
collection was set to peak-based detection using
the Agilent 1100 diode array detector with
timetable and peak settings as shown in Figure 1.
CA) under nonreducing conditions. Proteins were
visualized by Coomassie Blue staining.
Buffer Exchange with Spin Concentrators
To resolve bound fractions on 1D gels, fractions
were placed in 4-mL spin concentrators (Agilent
part number 5185-5991), with a 5 kDa molecular
weight cutoff (MWCO), and filled to maximum
volume with Buffer A. The sample was centrifuged
at 7,500 × g for 20–25 minutes at 8 °C. This process
was repeated three times for complete buffer
exchange. Buffer-exchanged samples were placed
in a clean Eppendorf tube for storage. Protein concentration for crude serum, flow-through fractions
and bound fractions (buffer exchanged) were
determined using a Pierce BCA protein assay kit.
Protein Analysis by SDS-PAGE
10
15
20
25
min
Dog
14.333
10
min
Monkey
10
6.062
4.037
Figure 2.
5
25
13.125
5
DAD1 A, Sig=280, 16 Ref=360, 100 (030304\DOG2.D)
0
20
14.316
3.965
0
mAU
2000
1500
1000
500
0
15
21.686
5
DAD1 A, Sig=280, 16 Ref=360, 100 (MONKEY1.D)
21.691
0
mAU
2000
1500
1000
500
0
Human
Bound
fraction
14.321
4.083
Flowthrough
fraction
Representative chromatograms for three of the six
species tested (human, monkey, and dog) using the
4.6 mm × 100-mm Multiple Affinity Removal
column are shown in Figure 2. The chromatograms
for monkey and dog serum resemble the human
serum chromatogram. The relative peak heights of
the flow-through fraction are small compared to
the bound fraction, which represents about
85%–90% of total protein, and is comprised of the
targeted six high-abundant proteins. The chromatograms for swine, rat, and mouse serum samples showed very large flow-through peaks under
the same conditions (data not shown), indicating
that their proteins do not bind well. The injection
volume for all species was held constant at 150 µL
and used an injector program.
21.680
DAD1 A, Sig=280, 16 Ref=360, 100 (HUMAN1.D)
13.072
mAU
2000
1500
1000
500
0
13.115
The protein patterns of serum samples before and
after depletion were visualized on SDS-PAGE gels.
The bound fractions were buffer-exchanged three
times into Buffer A; this minimized sample manipulation of the low-abundant flow-through fraction.
Equal amounts of protein mass from each fraction
and from crude serum were resolved in Novex
4%–20% Tris-Glycine gels (Invitrogen, Carlsbad,
Results and Discussion
15
20
25
min
Chromatograms of human, monkey, and dog serum on the Multiple Affinity Removal column. Crude
serum was diluted five-fold with Buffer A and filtered before loading onto the column. The sample
was loaded at a flow rate of 0.5 mL/min with Buffer A and allowed to run for 10 minutes. The bound
fractions were eluted with Buffer B (100%) for 7 minutes followed by re-equilibration for 11 minutes
with 100% of Buffer A.
3
The bound fractions were buffer-exchanged into
Buffer A and total protein content was measured
in all six species. Table 1 shows relative protein
removal as determined by protein content in crude
serum, flow-through fraction, and bound fraction.
The human and monkey samples showed approximately 85% depletion of total protein, whereas dog
serum showed 81% total protein depletion. The
levels of depletion in swine, rat, and mouse varied
at 42%, 23%, and 35% depletion respectively. Protein concentration of crude serum and flowthrough fractions were measured using the Pierce
BCA Protein assay kit. Percent depletion was
determined by determining concentration of the
flow-through fraction and the original crude
serum.
Table 1.
Human
1
2
3
Monkey
4
5
6
7
Relative Total Protein Removal for Six Species
Species
% Relative protein depletion
Human
85
Monkey
84
Dog
81
Swine
42
Mouse
35
Rat
23
When analyzed on SDS-PAGE, monkey and human
samples showed remarkable similarities in the gel
profile of crude serum and after depletion of the
targeted proteins (Figure 3). The dog serum data
indicates depletion of the major proteins targeted,
with the exception of transferrin (Figure 4). The
other samples, swine, rat, and mouse, showed
some level of depletion of the six high-abundant
proteins, but not complete removal (data not
shown).
Analysis of the targeted high-abundant protein
fractions was performed on the bound fractions for
all species. Samples were buffer-exchanged using
the Agilent Spin Concentrator with a 5 kDa MWCO
(Agilent part number 5185-5991), followed by separation by SDS-PAGE. Bands were excised and
4
subjected to in-gel tryptic digestion with reduction
and alkylation. Tryptic fragments were analyzed by
liquid chromatography/mass spectrometry
(LC/MS) and separated on a Zorbax 300SB-C18
column on an Agilent 1100 HPLC and Agilent SL
Ion Trap Mass Spectrometer. The results indicate
that the targeted proteins were being removed, as
expected, with albumin, IgG, IgA, haptoglobin,
transferrin, and antitrypsin being identified
repeatedly in all samples, as determined by Agilent
Spectrum Mill software (data not shown).
IgG
Albumin
Figure 3.
Comparison of human serum and monkey serum
after depletion of the targeted six high-abundant
proteins. Equal amounts of protein (5 µg) from each
fraction were separated by 4%–20% SDS-PAGE
under nonreducing conditions and visualized by
Coomassie Blue staining. Lane 1 - Molecular weight
markers; Lanes 2-4 are for human serum samples.
Lane 2 - Crude serum; Lane 3 - Low-abundant proteins (flow-through); Lane 4 - Bound proteins;
Lanes 5-7 are for monkey serum. Lane 5 - Crude
serum; Lane 6 - Low-abundant proteins (flowthrough); Lane 7 - Bound proteins (high-abundant
proteins). The arrows provide a reference showing
protein bands identified as IgG and albumin.
Dog serum
1
2
3
4
IgG
Transferrin
Albumin
Figure 4.
Enhanced detection by SDS-PAGE of low-abundant
proteins in dog serum after depletion with the
Agilent Multiple Affinity Removal column for human
serum. Lane 1 - Molecular weight markers;
Lane 2 - Crude serum; Lane 3 - Low-abundant proteins (flow-through); Lane 4 - Bound proteins (highabundant proteins). The arrows provide a reference
showing the location of identified proteins including
IgG and albumin in the depleted sample and transferrin in the flow-through fraction.
Conclusions
The Agilent Multiple Affinity Removal System
shows remarkable specificity for six high-abundant
proteins in human serum. When tested across several species including monkey (Rhesus monkey),
canine, swine, rat, and mouse, the columns display
varying levels of removal for the six targeted highabundant proteins. For monkey serum, depletion
of targeted proteins was observed, resulting in an
overall depletion as measured by protein concentration of about 84% of the protein mass, similar to
human serum samples. This is not surprising since
there is a great degree of homology between
human and monkey. For canine serum, depletion
of all targeted proteins was observed with the
exception of transferrin, with an overall depletion
of about 80% of the total protein from crude
serum. Overall, the Agilent Multiple Affinity
Removal System depletes greater than 98%–99% of
total targeted proteins (about 85% of the total protein mass in human serum is derived from these
targeted proteins) in human. The total protein
depletion is similar for monkey serum and removes
all targeted proteins except transferrin in dog
serum. The results for mouse, rat, and swine had
some high-abundant protein removal, but gel electrophoresis of the eluted proteins showed that
there was still high-abundant protein in each flowthrough fraction. The results of this study show
that the specificity and selectivity of the Multiple
Affinity Removal System is uniquely optimized for
human proteins and is consistent with previous
studies showing the highest possible selectivity for
the targeted proteins and low interaction with
nontargeted proteins.
References
1. K. Zhang et al. Agilent Multiple Affinity Removal
System for the depletion of high-abundant
proteins from human serum. Agilent
Technologies, publication 5988-9813EN.
www.agilent.com/chem/affinity
2. K. Zhang et al. Binding Capacity Assessment
and Optimization for the Multiple Affinity
Removal System. Agilent Technologies,
publication 5989-0601EN.
www.agilent.com/chem/affinity
For More Information
For more information on our products and services,
visit our Web site at www.agilent.com/chem.
William Barrett is a Proteomics Application Scientist at Agilent Technologies in Wilmington, DE, USA.
Correspondance: Dr. William Barrett
Email: [email protected]
5
www.agilent.com/chem
Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.
Information, descriptions, and specifications in this publication are subject to change
without notice.
© Agilent Technologies, Inc. 2004
Printed in the USA
April 14, 2004
5989-0957EN