Application Note

PROTEOMICS M E T A B O L O M I C S G E N O M I C S I N F O R M A T I C SG LY I L E VA L C Y S G L U G L N ALASERLEUASPARGCYSVALLYSPROLY S P H E T Y R T H R L E U H I S LY S

A Novel High-Capacity Affinity Column
for the Removal of the Top Seven
Abundant Proteins from Human PlasmaAuthors
Peter Mrozinski, Nina Zolotarjova,
Haiying Chen, James Martosella,and Hongbin Liu
Agilent Technologies, Inc.
2850 Centerville RoadWilmington, DE 19808-1610
USAAbstract
Recent HUPO studies indicate that plasma is becoming the preferred sample forproteome analysis over serum. Plasma is a unique sample, possessing proteinsthat span the entire human proteome. Analysis of plasma can provide valuable
information for the discovery of new biomarkers and novel drug targets.
However, the tremendous complexity of the plasma proteome presents extreme
analytical challenges in proteome characterization.
High-abundant proteins such as albumin, IgG, transferrin, haptoglobin, IgA, antitrypsin and fibrinogen comprise up to 90% of the total protein mass in plasma.
These high-abundant protein components interfere with the identification andcharacterization of important low-abundant proteins by limiting the dynamic
range of mass spectral and electrophoretic analyses.
Here we present data on extending this approach to a plasma sample to remove
the top 7 high-abundant proteins in plasma. The novel affinity-based resin
possesses a high capacity to deplete HSA, transferrin, haptoglobin, IgA, IgG,
anti-trypsin and fibrinogen from human plasma. Analysis of the flowthroughfractions during multiple runs indicates reproducibility and robustness of thedepletion process.
For the direct analysis of lower abundant proteins, the flowthrough fraction was
simultaneously desalted and fractionated on the Agilent macroporous Reversed
Phase C18 (mRP-C18) protein separation column. The combination of immunoaffinity depletion with high recovery RP-HPLC fractionation and subsequent
peptide analysis by HPLC-Chip/MS expanded the dynamic range of proteomic
analysis and allowed the identification of low-abundant proteins.

M E T I L E V A L C Y S G L U PROTEOMICS

Introduction
Human plasma continues to serve as the preferred sample
for the identification of marker proteins in drug development
and disease analysis. Due to the large dynamic range of theplasma proteome, identification of low abundance proteinsis a difficult task. Historically, one to two diagnostic protein
markers have been identified from plasma samples each year,
however, this number has actually declined to near zero inrecent years due to the limitations imposed by sample
analysis. In spite of the ease of solubility for most plasma
proteins, the vast number of associated proteins coupled withthe enormous dynamic range makes identification of low
abundance targets virtually unachievable using traditional
methods. Approximately half of the total protein mass inplasma is a single protein (albumin), with 10 additional
proteins comprising 90% of the total.
Previous reports have addressed the effectiveness of using
a high-capacity, multiple affinity removal column to deplete
both plasma and serum samples of interfering high abundanceproteins (1, 2, 3). This study examined a significant
enhancement in the current affinity column technology,
allowing plasma to be depleted of the top 7 high abundanceproteins. The new column had the same specificity,reproducibility, and high capacity as the industry standard
Top-6 column for serum, depleting >98–99% of the seventargeted proteins reproducibly for over 200 runs. The variability
in plasma samples, such as lyophilized versus fresh frozen
plasma, results in >95–99% removal of fibrinogen comparedto the remaining six proteins.
The removal of high abundance proteins improved the loading
capacity for 2DGE and LC/MS analyses, simplifying a complex
system for biomarker discovery. LC/MS/MS analyses confirmedthat the Plasma-7 column was superior to current affinity
depletion technologies, yielding highly specific protein
depletion. In addition, use of the column enabled a more
uniform comparison of plasma and serum samples,post-depletion.

2The enhanced depletion protocol resulted in an improved
dynamic range for proteomic analysis, thus facilitating thedetection and characterization of target marker proteins.
Agilent’s Multiple Affinity Removal approach with thePlasma-7 column provides a valuable commodity in thesearch for novel biomarkers and drug development.
Specific removal of seven high-abundant proteins depletes
approximately 85–90% of total protein mass from humanplasma. The low-abundant proteins in the flowthroughfractions can be studied. Removal of high-abundant proteinsenables improved resolution and dynamic range for onedimensional gel electrophoresis (1DGE), two-dimensional gel
electrophoresis (2DGE) and liquid chromatography/mass
spectrometry (LC/MS). The collected flowthrough fractions
may need to be concentrated depending on the downstream
applications

Experimental
Affinity column
The newly developed column is an extension and improvementon the Agilent Multiple Affinity Removal approach described
previously (1–3). Column contains newly developed affibody
ligands (Affibody, Sweden) specific for human fibrinogen.
The dimeric design of the Affibody® molecule is coupled with
Agilent’s proprietary methods to create the highest capacity
multiple depletion system. The column requires a two-buffer
system for operation. Buffers A and B are optimized tominimize co-adsorption of non-targeted proteins and to ensure
reproducibility of column performance and long column
lifetime. Buffer A—a salt-containing neutral buffer, pH 7.4,
used for loading, washing and re-equilibrating the column andBuffer B—a low pH urea buffer used for eluting the boundhigh-abundant proteins from the column.
Sample preparation
Before injection onto a high capacity multiple affinity column,
lyophilized, citrated human plasma (Sigma Cat. No. P9523)
was diluted four fold with Buffer A. The sample was transferred
to a 0.22 µm spin tube and centrifuged for 1 min. at 16,000 x g
to remove particulates. Diluted plasma was kept at 4°C.

Application NoteA Novel High-Capacity Affinity Column for the Removalof the Top Seven Abundant Proteins from Human Plasma

ELISA analysis of the flowthrough fraction
Standard sandwich enzyme-linked immunosorbent assays(ELISA) were used to determine the completeness of removalof targeted proteins from human plasma.

The dried fractions from the mRP column were digested usinga trifluoroethanol-based protocol (5). Peptides were analyzed
by 2D nanoflow LC/MS/MS on an Agilent HPLC-Chip/MS
system interfaced with an Agilent XCT Ultra Trap LC/MS.

Briefly, assay plates were coated with 100 µl of flowthroughfraction proteins or plasma samples diluted 10x in buffer A.
After an overnight incubation at 4°C, plates were washed with
PBS and the nonspecific binding sites were blocked with
200 µl of blocker solution (Bio-Rad) for 2 hours. After washingplates with PBS, 100 µl of affinity-purified rabbit anti-humanantigen antibodies were added in blocker solution. Plates wereincubated for 2 hours, washed, a secondary antibody HRPconjugated goat anti-rabbit IgG (Sigma) was added for 1 hour.
After washing with PBS, liquid substrate (TMB) was added
and the absorbance was measured at 655 nm.

• Salt Steps: sample (0), 20, 50, 100, 150, 200, 250, 350, 500,700, 1000, 2000 mM ammonium acetate.

SDS-PAGE
Flowthrough fraction proteins were loaded on the SDS-PAGE
without manipulations. Bound fractions were pH adjusted andloaded onto gels. Protein concentrations were analyzed using
a BCA protein assay kit (Pierce). Samples were stored at–70°C until analysis. SDS-PAGE analysis was carried out using
Invitrogen Tris-Glycine pre-cast gels (4–20% acrylamide,10 wells, 1 mm). Proteins were visualized by Coomassie Bluestaining with GelCode Blue (Pierce).

The Agilent Plasma-7 Multiple Affinity Removal Column
presents significant advantages over previous methods of low
abundance protein analysis by enabling the easy removal of
the top seven proteins (transferrin, antitrypsin, IgA, IgG,
haptoglobin, and fibrinogen) from crude samples of humanplasma. The removal of highly abundant proteins reduces thedepth of dynamic range within plasma samples, thus enablingan increased loading capacity for proteins found in much
lower abundance for PAGE and LC/MS/MS analyses.

mRP protein fractionation
The flowthrough fraction was directly separated on amacroporous Reversed Phase (mRP-C18) protein column(Agilent) after denaturation with urea (4). 24 fractions werecollected and dried with a Speed-Vac concentrator.

To determine loading capacity limits for the Plasma-7 column,
sequential injections of increasing plasma volumes were madeand the flowthrough fractions were separated using PAGE
and Coomassie Blue staining. Results show that even at
the highest plasma injection volume (80 µL), there was nobreakthrough of targeted proteins evidenced by staining(Figure 1; Lanes 3–5). The absence of breakthrough proteinsfrom an injection of 80 µL of crude plasma demonstrates thedramatic improvement in column loading capacity for thissystem as compared to other existing high-affinity columns.
In addition, subsequent ELISA analysis reported a 99.4%
depletion of fibrinogen from all three plasma loads, confirming
the effectiveness of target protein depletion (data not shown).

LC/MS/MS analysis
To analyze the specificity of the immunodepletion, the boundfraction was resolved by SDS-PAGE. Coomassie-stained
protein bands were cut and processed with the Agilent
Technologies Protein In-gel Tryptic Digestion Kit. Peptides
were analyzed by LC/MS/MS on an Agilent HPLC-Chip/MS
system interfaced with an Agilent XCT Ultra Trap LC/MS.
Results were processed by Agilent Spectrum Mill software.

www.agilent.com/chem/proteomics• RP Mobile Phase: H2O w/0.1% FA, 3% ACN & ACN w/
0.1% FA – 500 nL/min• Enrichment Mobile Phase: H2O w/0.1% FA, 0.05% TFA &3% ACN – 4 µL/min• RP Gradient: 3% B @ 0 min., 10% B @ 2 min., 35% B @20 min., 50% B @ 22 min., 95% B @ 22.5 min.,95% B @23.5 min., 3% B @ 24 min.

Results and Discussion

3M E T I L E V A L C Y S G L U PROTEOMICSFigure 1. 1D-gel of bound and flowthrough fractions from a
4.6 x 100 mm Plasma-7 Multiple Affinity Removal Column.
Lane 1 – Mark12 Standard; Lane 2 – Human Plasma;
Lane 3 – Flowthrough (60 mL); Lane 4 – Flowthrough(70 mL); Lane 5 - Flowthrough (80 mL); Lane 6 – Boundfraction (10 mg). Boxed bands show respective regions ofthe gel (Gel bands A–P) that were excised and used in theidentification of bound proteins.

To identify the proteins that were removed from plasma,proteins representing the bound fraction from the Plasma-7
Multiple Affinity Removal Column were separated by PAGE,
extracted using in-gel trypsin digestion, and analyzed by
LC/MS/MS (see Lane 6 of Figure 1). Of the 16 gel bands
analyzed for the bound protein fraction, 13 contained proteinstargeted for removal (Table 1). Other proteins removed
included Complement C3 and B, two apolipoproteins, and thehemoglobin alpha chain. These results confirm the highspecificity of the Plasma-7 column for removal of targeted
proteins. When ELISA and LC/MS/MS results are viewed inunison, it is obvious that the Plasma-7 column offers
comprehensive depletion of targeted high abundance proteins,while allowing lower abundance proteins to be collected forseparate downstream analysis.

Table 1. Protein identification based upon LC/MS/MS
analysis of excised, bound fraction gel bands
Gel bandProtein identified*A

Fibrinogen

BFibrinogen, Haptoglobin, HSACIgG, IgA, Complement C3

DIgG, HSA, IgA

EIgG, HSAF

IgG, HSA, Ceruloplasmin

GHSA, Haptoglobin, IgG

HHSA, Complement B preI

IgG, HSA

JTransferrin, HSA

KHSA, Transferrin, alpha-1-antichymotrypsin

LAlpha-1-antitrypsin, HSAM

HSA

N

Apolipoprotein HO

Apolipoprotein

PHemoglobin alpha chain*Items in italics are untargeted proteins removed non-quantitatively.

4Application Note

In addition to ensuring complete separation of proteins, theHuman Plasma-7 column is extremely robust, performing
reproducibly over 200 runs. When column chromatograms
were compared over 200 runs (at checkpoints every 40 runs),
there was very little change in peak dimensions (Figure 2). The
consistency of separation and repeatability over hundreds ofruns guarantees high quality results for low abundancetargets.

A Novel High-Capacity Affinity Column for the Removalof the Top Seven Abundant Proteins from Human Plasma

The reproducibility of the Human Plasma-7 column isdemonstrated in further SDS-PAGE analysis of the flowthroughfractions (Figure 3). SDS-PAGE analysis of the flowthroughfractions from 200 runs on this column shows consistent
results from beginning to end. Flowthrough samples wereequally loaded based on protein concentration (10 µg per well)and exhibited no change in protein banding pattern for any ofthe 200 runs.

Figure 2. LC chromatograms during 200 runs of affinity removal using the Plasma-7 column for high abundance protein removal from crude plasma samples.

www.agilent.com/chem/proteomics

5

M E T I L E V A L C Y S G L U PROTEOMICS

A partial representation of the numerous low-abundant
proteins found in the flowthrough fraction as analyzed by
LC/MS/MS is shown in Table 2. Multiple unique proteins wereidentified from the flowthrough fraction; many proteins were
important precursors to signal transduction pathway
components. Others included potential protein regulators suchas nuclear receptor corepressors, serine/threonine-protein
kinases, and protein kinase C-like structures.

Conclusions

Figure 3. Analysis of column performance during 200 runs as visualizedusing SDS-PAGE separation. Lane 1 – Human Plasma; Lane 2 – Mark12
Standard; Lane 3 – Flowthrough (Run 1); Lane 4 – Flowthrough (Run 40); Lane
5 – Flowthrough (Run 80); Lane 6 – Flowthrough (Run 120); Lane 7 –Flowthrough (Run 160); Lane 8 – Flowthrough (Run 200). A total of 10 µg ofprotein was loaded into each well.

6The Human Plasma-7 column from Agilent offers a robust andconsistent tool, particularly useful to those requiring
heightened sensitivity for proteomic discovery through theremoval of high abundance proteins. In addition, thecombination of immunoaffinity chromatography with highrecovery RP-HPLC fractionation and subsequent HPLC-Chip/
MS peptide analysis expands the dynamic range by enabling
the identification of low abundance plasma proteins. As one ofthe highest capacity columns available, the Human Plasma-7
offers an accurate and dependable high-throughput method forprotein separation.

Application Note

A Novel High-Capacity Affinity Column for the Removalof the Top Seven Abundant Proteins from Human Plasma

Table 2. Partial representation of the numerous low abundance proteins identified using direct 4D LC/MS
analysis of the flowthrough fraction
ProteinAccession No.

No. distinct peptides

Hepatocyte growth factor-like protein precursor (ng/mL)

P269277Properdin precursor (Factor P)

P27918

6Fetuin-B precursor

Q9UGM5

7Hepatocyte growth factor activator precursor

Q04756

6L-selectin precursor

P14151

6Insulin-like growth factor binding protein 3 precursor (ng/mL)

P17936

5

Selenoprotein P precursor

P49908

5

Monocyte differentiation antigen CD14 precursor

P08571

4

Insulin-like growth factor II precursor (ng/mL)

P07456

3

Platelet basic protein precursor (ng/mL)

P02775

3Vascular endothelial-cadherin precursor (ng/mL)

P33151

3Lipopolysaccharide-binding protein precursor

P184283

Neutrophil defensin 3 precursor

P59666

3

Serum amyloid A protein precursor

P02735

3

Intercellular adhesion molecule-2 precursor

P135982

Ryanodine receptor 1P11716

3

EGF-containing fibulin-like extracellular matrix protein 1 precursor

Q12805

2

CD44 antigen precursor

P160702

Serine/threonine-protein kinase

Q005362

Glutamate receptor 1 precursor

P42261

2

Sentrin-specific protease 2

Q9HC622Nesprin 2Q8WXH0

2

Nuclear receptor corepressor 1Q60974

2Protein kinase C-like 2

Q165132

www.agilent.com/chem/proteomics

7

Application NoteM E T I L E V A L C Y S G L U PROTEOMICS

References
1. Szafranski, C., Bailey, J., Turck, C.W., Nicol, G., Martosella, J. and Zolotarjova,
N. (September 2004) Enhancing analytical access to low-abundant proteinsin the human plasma proteome. PharmaGenomics:40–46.
2. Bjorhall, K., Miliotis, T. and Davidsson, P. (2005) Comparison of different
depletion strategies for improved resolution in proteomic analysis of humanserum samples. Proteomics. 5:307–317.
3. Chromy, B.A., Gonzales, A.D., Perkins, J., Choi, M.W., Corzett, M.H., Chang,B., Corzett, C.H. and McCutchen-Maloney, S.L. (2004) Proteomic analysis ofhuman serum by two-dimensional differential gel electrophoresis after
depletion of high-abundant proteins. Journal of Proteome Research.
3:1120–1127.
4. Martosella, J., Zolotarjova, N., Liu, H., Nicol, G. and Boyes, B.E. (2005)
Reversed-Phase High-Performance Liquid Chromatographic Prefractionationof Immunodepleted Human Serum Proteins to Enahnce Mass Spectrometry
Identification of Lower-Abundant Proteins. Journal of Proteome Research.
4:1522–1537.
5. Agilent Nanospray and HPLC-Chip/MS Protein Identification Solutions.
G2458-90003:67–78.

About Agilent’s Integrated Biology Solutions
Agilent Technologies is a leading supplier of life science
research systems that enable scientists to understandcomplex biological processes, determine diseasemechanisms, and speed drug discovery. Engineered forsensitivity, reproducibility, and workflow productivity,
Agilent's integrated biology solutions include
instrumentation, microfluidics, software, microarrays,
consumables, and services for genomics, proteomics, andmetabolomics applications.

Learn more:
www.agilent.com/chem/proteomics
Buy online:
www.agilent.com/chem/store
Find an Agilent customer center in your country:
www.agilent.com/chem/contactus
U.S. and Canada
1-800-227-9770
agilent_inquiries@agilent.com
Europeinfo_agilent@agilent.com
Asia Pacificadinquiry_aplsca@agilent.com
Research use only. Information, descriptions, and specifications in thispublication are subject to change without notice.
Agilent Technologies shall not be liable for errors contained herein or forincidental or consequential damages in connection with the furnishing,performance or use of this material.
© Agilent Technologies, Inc. 2006
Printed in the U.S.A. September 11, 2006
5989-5640EN