Comparative Analysis of Control and Breast
Cancer Sera Using a Multidimensional
Separation Strategy
Application

Proteomics

Authors
Haiying Chen, Nina Zolotarjova, and Hongbin Liu
Agilent Technologies, Inc.
2850 Centerville Road
Wilmington, DE 19808-1610
USAAbstract
Human serum is frequently used for the discovery of new
disease markers, drug targets, or studying protein expression patterns in human biology. However, the presence ofhigh-abundance proteins in serum interferes with theanalysis of low-abundance proteins by limiting thedynamic range of mass spectral, electrophoretic, andchromatographic analysis. Reducing sample complexity
becomes an important first step in proteomic analysis ofcomplex samples and successful biomarker discovery.
We applied a new multidimensional separation techniqueto reduce sample complexity in the comparative analysisof serum samples from breast cancer patients and healthy
control subjects.

for depleted serum samples were greater than 99%.
Reproducibility among patient samples was excellent based on the UV trace overlays. The mRP-C18
fractionated serum samples were further analyzed
by 1D-PAGE and followed by in-gel tryptic fragmentation and peptide analysis using matrixassisted laser desorption ionization-time of flight(MALDI-TOF) for selected gel bands. The mRP-C18
fractions were also analyzed by in-solution digestion and two dimensional-liquid chromatography
tandem mass spectrometry (2D-LC/MS/MS). The
combination of affinity chromatography with highrecovery mRP-C18 fractionation and subsequentgel separation reduces sample complexity andgreatly increases the number of proteins identified. This multidimensional separation enables theidentification of differences in protein expression
between healthy and breast cancer serum samples.
This new strategy can be adapted to otherbiological analysis and cancer model studies.

ExperimentalSera

Introduction
A high capacity multiple affinity removal column isapplied to specifically remove six high-abundant
proteins (albumin, IgG, transferrin, haptoglobin,IgA, and anti-trypsin) in human serum. A macroporous reverse-phase C18 (mRP-C18) column wasused to fractionate the depleted serum. Under theoptimized mRP-HPLC condition, protein recoveries

Breast Cancer and Healthy control sera were purchased from Genomics Collaborative (Cambridge,MA).
High Capacity Multiple Affinity Column
An Agilent High Capacity Multiple Affinity
Removal Column (Hu6-HC), 4.6-mm id × 50 mm(Agilent part number 5188-5332) was used toremove the top six proteins from the cancer andcontrol sera. The column was used according to
manufacturer’s published protocol.
Briefly, serum was diluted 5× with Buffer A,loaded onto the column, and the flow-through collected, pooled, and concentrated. Buffer B wasused to elute the bound proteins from the column
prior to requilibration in Buffer A.
Protein Fractionation of Flow-through Proteins
The concentrated flow-through fractions from several runs of cancer or control were diluted in 6M
acidic urea. The urea denatures the proteins andaids in recovery while the acid prevents carbamylation at the elevated temperatures during separation. An mRP-C18 column, 5 µm, 9.4-mm id × 50 mm(Agilent specially packed and available as customorder, part number 5188-5345) was used to fractionate the proteins. The flow-through proteins
were separated under RP conditions on an Agilent
1100 HPLC. Forty-nine fractions were collectedunder optimized conditions and analyzed by
1D SDS-PAGE, and four fractions from each serum
sample were also analyzed by 2D-LC/MS/MS.

detection permits, on a broad scale, the ability tocompare chromatograms from control samples tothose of disease/treated samples for gross differences. This permits a more rapid screening for proteins of interest while all fractions are available foranalysis after the initial screen. A comparison ofbreast cancer serum to control serum is performed
here using the Multiple Affinity Removal Column
to fractionate high-abundant proteins prior toprotein fractionation with the mRP-C18 column.
Figure 1 shows the initial depletion as analyzed bySDS-PAGE gel. No discernable differences can bedetected between the control sample and thepatient samples from the initial depletion of thesix major abundant proteins in serum, including
albumin, IgG, IgA, transferrin, anti-trypsin, andhaptoglobin.

12345678

910SDS-PAGE
Aliquots from the flow-through and bound fractions were run on 4%–20% Tris-Glycine pre-castgels (Invitrogen). For bound fractions, samples
were desalted with 20-mM Tris-HCl. Sample preparation and electrophoresis conditions followed themanufacturer’s suggested protocol. Indicated gel
spots were cut and processed with Agilent in-gel
digestion kit (Agilent part number 5188-2749) andanalyzed by MALDI-TOF.
MS Analysis
LC/MS/MS: Protein digests were first fractionatedwith an SCX column. Each fraction was analyzed
with 1D LC/MS/MS using Agilent 300SB-C18,5 µm, 0.3-mm id × 5 mm as a trap column, and areverse-phase column SB C18, 3.5 µm, 0.075-mm id× 150 mm, with Agilent 1100 HPLC (300 nm), andMSD Trap XCT. Results were processed bySpectrum Mill software (Agilent Technologies).

Results and Discussion
The ability to fractionate samples at whole protein
levels aids in the reduction of sample complexity.
The process of RP-separation coupled to UV

2Figure 1.

SDS-PAGE analysis of the healthy control and twobreast cancer patients’ serum fractions before andafter immunodepletion. Lane 1, MW markers; Lane 2,control serum, SIW3TSB; Lane 3, control serum flowthrough fraction; Lane 4, patient 1, SIDV1SC6; Lane 5,patient 1, SIDV1SC6 flow-through fraction; Lane 6,patient 2, SIDV1S3W; Lane 7, patient 2, SIDV1S3W
flow-through fraction; Lane 8, control serum, boundfraction; Lane 9, patient 1 bound fraction; Lane 10,patient 2 bound fraction. A total of 8-µg protein wasloaded for each sample.

The flow-through fractions for each sample wereprepared according to the methods section prior toinjection and fractionation on the mRP-C18
column. The separation was performed at 80 °C
using a multistep gradient for optimal recoveryand resolution. The chromatograms were staggered
and compared for any gross changes. As indicatedin Figure 2, there were observable UV differences
between the control sample and the patient samples,particularly around fractions 19–28.
Gel-based and in-solution digests were used to analyze each gel band or four mRP-C18 fractions.
Fractions from the mRP-C18 column were first
analyzed on SDS-PAGE gels for differences inpatterns or staining/expression levels (Figure 3).
Various protein bands were labeled and excisedfrom the gel, digested and analyzed by LC/MS foridentification. In addition, an aliquot from four
fractions for control and patient that showed differences was subjected to in-solution tryptic
digestion.
Proteins that were identified from in-gel tryptic
digests are listed in Table 1. The completeness ofremoval of haptoglobin was determined to be morethan 99% by ELISA for normal control serum.

Haptoglobin is an acute-phase reactant and overexpressed in cancer patients. This is noted by thepresence of haptoglobin in the patient samples but
not in the control.
Table 1.

Proteins Identified from SDS-PAGE Gel Bandsby LC/MSGel band

Protein(s) identified

123456789101112131415

Histidine-rich glycoprotein
Kininogen
C4, Synaptonemal complex protein
Kininogen, Coagulation factor XIIIBHaptoglobin
Histidine-rich glycoprotein
Kininogen
Hemopexin
Serum Amyloid A
Complement C8Complement factor B
Complement C7
Clusterin
Complement C4Complement C4, Synaptonemal complex protein

Fractions 19–28 analyzed by
1D- and 2D-LC/MS/MS

Control

Patient 1

Patient 2

Figure 2.

The mRP-C18 chromatograms from control, patient 1, and patient 2 fractionations. The box indicates the fractions(19–28) chosen for initial analysis by LC/MS based on differences within the UV trace.

3Healthy control
KDKD

116

116

31
2

9766

9766

55
4255
4230

30

22
1722
176618192021222325

26

2728

29 30-31 32 33-34 35

Breast cancer patient 1KD

KD

14 15
567116
9766

4116

897
66

55
4255
4230

30
2222

917
6

17
1920

21

22

2325

26

2728

29 30-31 32 33-34 35

SDS-PAGE analysis of the control and patient mRP-C18 fractions. The numbered gel bands wereexcised, digested and identified by LC/MS (Table 1).

The analysis of four fractions from the mRP-C18separation revealed several distinct proteins that
only appeared in the control sample (Table 2), onlyappeared in the patient sample (Table 3), orappeared in both control and patient, though atdifferent levels of expression (Table 4). The results
indicate the power of coupling immunodepletion
to protein fractionation for the potential identification of biomarkers. The initial prescreen of the UV

413618Figure 3.

101112

chromatogram allows one to quickly focus on thegross differences before probing further with theremaining fraction. The ability to recover proteinsat >98% when using the mRP-C18 permits the comparison of the immunodepleted serums. Together,
the mRP-C18 and multiple affinity columns provide reliable, reproducible and robust performancefor biomarker discovery.

Table 2.

Proteins Identified in Healthy Control Serum Only

Protein names

NumSpectra*

Numpeps**Full-length cDNA clone CS0DD006YL02 of neuroblastoma

177

Complement C754

Slice isoform 2 of inter-a-trypsin inhibitor heavy chain H4 precursor

53

Tropomyosin 433

Complement C1r-like proteinase

62Protein names

NumSpectra*

Numpeps**Complement C4 precursor

167Splice isoform short of complement factor H-related protein 2 precursor

234Complement factor H-related 565

IGHM protein

85Splice form 2 of attractin precursor

44Clusterin

63Extracellullar matrix protein 1 precursor

83*Number of matched MS/MS spectra**Number of unique peptides

Table 3.

Proteins Identified in Cancer Patient 1 Serum Only

*Number of matched MS/MS spectra**Number of unique peptides

Table 4.

Proteins in Both Sera Samples with Different Expression Levels

Protein Names
Serum amyloid A protein precursorCystatin C precursor
Lumican precursor
Coagulation factor V precursor
Selenoprotein P precursor
Complement component C8 gamma chain precursor
AMBP protein precursor
Inter-alpha-trypsin inhibitor heavy chain H2 precursor
Hepatocyte growth factor activator precursor
Coagulation factor XIIIB chain precursor
Splice isoform 1 of complement factor H precursor
Plasma retinol-binding protein precursor

Healthy control
NumSpectra* (Numpep**)
8(4)
1(1)
2(1)
2(2)
1(1)
5(3)
9(4)
8(2)
6(2)
7(4)
260(40)80(5)Patient NumSpectra*(Numpep**)
107(8)
9(6)
8(4)
6(5)
3(2)
13(4)
21(6)
17(4)
12(4)
3(3)
98(28)
28(7)Ratio***
0.08
0.2
0.25
0.33
0.33
0.38
0.42
0.47
0.5
2.3
2.65
2.85

*Number of matched MS/MS spectra**Number of unique peptides***Ratio of control over Patient 1 spectra number5

www.agilent.com/chem

Conclusions
The high-capacity affinity removal column greatly
reduces serum complexity by removing the six
most-abundant proteins. The ability to fractionate
whole protein with the mRP-C18 column allowed
for excellent reproducibility and high protein
recoveries. The multidimensional separations
enable a thorough serum sample comparison fromhealthy and diseased subjects by 1D SDS-PAGE
and 2D-LC/MS/MS. A comparison of proteins identified in the control and patient sera indicates anumber of proteins with significant differential
expression levels which were identified in these
two samples. This multidimensional separation
strategy may be used to identify protein biomarkers critical to pathological events and pharmacologic intervention from a variety of sample sources.

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visit our Web site at www.agilent.com/chem.

Haiying Chen is a manufacturing engineer with the Integrated Biology Systems division of Agilent Technologies.
Nina Zolotarjova and Hongbin Liu are R&D scientists within the BioSeparationsgroup, Integrated Biology Systems of Agilent Technologies.

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. 2006
Printed in the USA
March 20, 2006
5989-4931EN