Comparative Analysis of Serum Samples
with the Agilent mRP-C18 Column - A New
Technology for Protein Fractionation
Application

Proteomics

Authors
William Barrett, James Martosella, Nina Zolotarjova,
Liang-Sheng Yang, Cory Szafranski, and Gordon Nicol
Agilent Technologies, Inc.
2850 Centerville Road
Wilmington, DE 19808-1610
USA

Abstract
Proteomic samples such as human serum are an extreme
challenge to study due to the large dynamic range of theproteins of interest. The proteins in human serum canrange from those considered “high-abundance” proteins,present at 2%–50% of the total protein mass (typically theµg/mL–mg/mL range) to those considered tissue biomarkers present in the pg/mL–ng/mL range. Multidimensional separation techniques have provided one way toaddress the complexity of these samples. A reversedphase (RP) HPLC method for the separation and fractionation of immunodepleted protein samples was developed
using a novel macroporous reversed-phase (mRP)
column. The optimized method and special column material combine to provide high-protein recoveries, reproducible fractionation, and higher column loads. Samplesof human serum, depleted of the six most abundant proteins, were separated on a novel mRP-C18 column resulting in high protein recovery, good protein resolution, andexcellent reproducibility. This allowed for a direct comparison of several immunodepleted human sera samples,
which showed differences in chromatographic UV

profiles, providing a quick screen of protein differences infractions that can then be analyzed by liquid
chromatography/mass spectrometry.

Introduction
The drive to discover novel protein serum biomarkers in either drug development or diseasemodels is not only vital to further research, but isalso a major challenge. The dynamic range of proteins in human serum spans greater than 10 ordersof magnitude with six proteins representing 85% of
the total protein mass, including albumin, IgG, IgA,
transferrin, haptoglobin, and alpha-1-antitrypsin.
Depletion of high-abundance proteins with theAgilent Multiple Affinity Removal System can provide access to previously undetectable proteins.
Typically, immunodepleted samples are further
fractionated with techniques such as 2-dimensionalgel electrophoresis (2DGE), multidimensionalliquid chromatography/mass spectrometry(LC/MS), or offline methods employing orthogonal
separation techniques. The fractionation strategies
continue to develop as scientists test for optimized
separations of proteins, which provide high recoveries, efficient separations, and reproducibility, allof which are required for the eventual validation ofprotein biomarkers. Currently, multi-dimensional
separations suffer from poor sample recoveries
and reproducibility. Lack of reproducibility associated with 2DGE often makes it difficult to compare
samples directly. In addition, protein recoveries
from RP chromatography typically range from30%–80% and are often not reproducible. The

ability to couple the Agilent Multiple Affinity
Removal System to deplete the top six highabundance proteins in serum with a RP system tofractionate proteins would enable researchers tosimplify serum samples and identify potential biomarkers. In this study, the protein fractionations
from three immunodepleted serum samples, a control, a cortisol-deficient sample, and a highrheumatoid factor sample were compared for differences. The high recovery and increased resolution enabled an improved comparative analysis ofserum samples. This technique allows one to
rapidly screen differences that can then be
identified by LC/MS.

separation. Approximate final concentrations were
6M urea and 1.0% acetic acid.
Samples of serum were depleted of the highabundance proteins using the Agilent Multiple
Affinity Removal Column as stated. Protein fractionation of the flow-through fraction from immunodepletion was performed on the 4.6 mm ID ×
50-mm mRP-C18 column (part number 5188-5231).
Sample preparation prior to loading on themRP-C18 column is necessary for resolution andreproducibility and contributes to the highrecovery of low-abundance proteins.
LC Fractionation System

Experimental
The Multiple Affinity Removal System for removing
albumin, transferrin, IgG, IgA, haptoglobin andantitrypsin from human serum is a product from
Agilent Technologies (Wilmington, DE). A 4.6-mm
× 100-mm Multiple Affinity Removal column (partnumber 5185-5985) was used with a mobile phasereagent kit (Agilent Technologies, part number
5185-5986). Sample loading, washing, and column
regeneration is done using Buffer A, and for boundprotein elution Buffer B is used according to manufacturer protocols. Injections of diluted serum
were performed according to manufacturer protocols for a 4.6-mm × 100-mm column. The flowthrough fractions were automatically collected bytime into 1.5-mL plastic tubes (part number
5188-5251) using an Agilent 1100 HPLC equippedwith a thermostatted analytical-scale fraction collector. The depleted serum samples containing lowabundance proteins in the flow-through fractions
were collected and stored at –20 °C until analysis.
A sample of serum from a healthy control subject,cortisol-deficient sera (Sigma number 7269), andsera with high rheumatoid factor (Sigma number3145) were all immunodepleted to remove six highabundance proteins as per manufacturer’s
protocol.

An Agilent 1100 HPLC equipped with a binary
pump, thermostatted autosampler, thermostatted
column compartment, and a thermostatted analytical-scale fraction collector was used for fractionating the samples. Due to the larger volume requiredfor complete injection of the samples, the standard
100-µL needle and sample loop from the autosampler was replaced with a 900-µL needle andmetering device (Agilent part number G1363A).
Note: The use of multidraw is not recommended or
supported due to decreased peak shape and large
delay volume.
Repeat Inject Method for Large Volume Loading
For loading sample volumes greater than 900 µL,
an isocratic method (Figure 1) and sequence table
is required. The method involves the injection of adesired volume based on total sample volume followed by an isocratic run of 3 minutes to load themRP-C18 column. This can be repeated several
times for larger volumes using the sequence table
until the final injection, which would be doneusing the optimized separation method (Figure 2).

Sample Preparation of Immunodepleted Serum for
Loading onto the mRP-C18 Column
The flow-through fractions from each immunodepleted serum were collected, and protein concentrations determined via the BCA Protein Assay(Pierce, Rockford, IL). Total protein (300 µg) fromeach flow-through fraction in approximately 1 mL
was prepared for direct-loading onto the mRP-C18
column. Into each sample, 0.48 g of urea pellets
was dissolved and 13 µL of neat glacial acetic acidadded to acidify the samples and prevent carbamylation at the elevated temperature required for the2

Figure 1.

Example Agilent 1100 set-up screen for isocratic
loading of large sample volumes.

Figure 2.

Sequence table to load large sample volumes; the example demonstrates two isocratic
injections of 500 µL followed by the last injection that runs the gradient elution.

Protein Fractionation
After sample preparation with urea and glacialacetic acid, the immunodepleted serum was separated under RP conditions using a linear multisegment gradient (Table 1). Fraction collection was
performed by time, collecting 1-minute time slices
starting at 1 minute and continuing to 53 minutes.
Table 1.

Protein Fractionation Linear Multisegment Gradient

Flow
Stoptime
Posttime
Column temperature
Detection
Starting Solvent Composition
Solvent A
Solvent B
Pressure limit
Gradient

0.75 mL/min
58 min20 min
80 °C
UV 280 nm

gradient typically employed for desalting and concentrating the flow-through fraction (Table 2).
Fractions were collected and pooled and thesample dried using a SpeedVac. The column wasthen placed inline and several injections were performed for each serum sample and the fractions
collected, pooled and dried as above. The pellets
were resolubilized in 3M urea/1% Triton X-100
detergent solution. Protein concentrations were
determined using a BCA protein assay kit (Pierce,
Rockford, IL). Recovery data for the sample withand without the column installed were used todetermine accurately the absolute protein
recoveries.
Table 2.

97% (Water/0.1% TFA)
3% (ACN/0.08% TFA)
200 barTime (min)
%B
0
3
1
3
6
30
39
55
49
100
53
100
58
3

Recovery Conditions
To measure protein recovery, several injections ofsera (approximately 47 µg) were performed without the column installed inline using a fast

Protein Desalting/Concentration Gradient

Flow
Stoptime
Posttime
Column temperature
Detection
Starting Solvent Composition
Solvent A
Solvent B
Pressure limit
Gradient

0.75 mL/min
13 min10 min
80 °C
UV 280 nm
97% (Water/0.1% TFA)
3% (ACN/0.08% TFA)
250 barTime (min)
%B
0
10
3
10
5
70
6
100
8
100
10
10

3

Tryptic Digest of Protein Fractions

Results and Discussion

The selected mRP-C18 column fractions were resuspended in 25 µL of 50-mM NH4HCO3 and 25 µL of
trifluoroethylene (TFE). Then 3 µL of 200-mM
dithiothreitol (DTT) was added and the sample was
mixed briefly on a vortex mixer. The solution was
incubated at 60 °C for 1 hour. The reduced proteins
were alkylated with iodoacetamide (IAM) by adding
3 µL of a 200 mM IAM solution followed by incubation in the dark at room temperature for 1 hour.
After incubation, 3 µL of 200 mM DTT was added toreact with the excess IAM.

The ability to reduce the complexity of proteomic
samples typically involves orthogonal techniquessuch as 2D-chromatography or the use of gel electrophoresis. The Agilent mRP-C18 column provides
a tool to reduce complexity at the protein level
prior to downstream analysis. The mRP-C18
column offers several advantages over standard
reversed phase columns including high recovery,
reproducibility, and resolution. The recovery froma column is critical in the analysis of samples toprevent cross-contamination due to carryoversince typical recoveries in RP HPLC methods canrange from 30%–80%. Recovery is also important in
the validation of biomarkers by allowing for a
direct quantification experiment between control
and treated/ diseased samples, where inefficient
recovery from typical RP HPLC methods prohibits
its use in biomarker validation.

Prior to trypsin digestion, the TFE concentration in
the solution was adjusted with 50 mM NH4HCO3 toless than 5%. Trypsin was added at a ratio of
approximately 50:1 protein to enzyme and proteins
were digested overnight at 37 °C. An aliquot wasused for LC/MS analysis.
LC/MS Analysis
Digested fractions from the mRP-C18 column wereanalyzed by LC/MS on an Agilent 1100 MSD Trap
SL. A trapping column (Agilent 300SB-C18, 5 µm,
0.5 mm × 35-mm column) was used to capture andconcentrate the sample peptides prior to separation
on a RP column (ZORBAX 300SB-C18 5 µm, 0.3 mm
× 150 mm, Agilent Technologies). The flow rate for
the RP separation was 5 µL/min. Peptides weretrapped on the trapping column, which was thenswitched inline with the flow for the analytical RP
column, and the peptides were eluted off the trap
column and onto the RP column. The gradient for
the RP and salt slices is as follows:
Time (min)
%B

0
5

5
5

50
40

55
70

58
70

60
5

Spectrum Mill software was used for data analysis.
The following filters were used for peptide and protein identification: peptide score 9, protein score 11
and %SPI: 70%. Only fully tryptic peptides wereconsidered with one tryptic missed cleavage
allowed.

4

Recovery data for samples that were obtained withand without the mRP-C18 column inline show 99%
recovery (Table 3).
Table 3.

Recovery of Serum Proteins from the Agilent
mRP-C18 Column as Determined by the BCA Assay

Protein conc.
no column(µg)

Protein conc.
mRP-C18
recovery (µg)% Recovery

49.8

49.3

99

*N=5. Percent recovery is calculated from the
mRP-C18 column recovery versus the fraction collected with no column.

A further examination of protein recovery was
performed by injecting and separating immunodepleted serum on the mRP-C18 column, followed bya blank injection. Analysis of the chromatograms
shows no significant UV absorbance for the blanksample suggesting greater than 99% recovery of theserum from the first injection (Figure 3). The
excellent recovery from the mRP-C18 column provides the ability to compare a diseased and control
sample without carryover concerns.

Immunodepleted serum

Blank

Figure 3.

Two chromatograms show the separation of immunodepleted serum followed by a blank injection.
The high recovery from the mRP-C18 column is demonstrated by the absence of significant
absorbance in the blank run.

Reproducibility would provide a measure of confidence to compare and validate potential protein
biomarkers identified in serum. The reproducibility of five immunodepleted serum injections isattained with the optimized method and temperature for protein fractionation as shown in Figure 4.
The inset demonstrates the robust reproducibilityof the Agilent mRP-C18 column for two immunodepleted serum samples that were injected 1 week
apart. The column was used extensively during theweek for many different serum samples.

Figure 4.

Reproducibility of the mRP-C18 column as shown in five consecutive injections. Inset, shows two injections ofserum performed 1 week apart.

5

Reproducibility, high recovery, and efficient resolution allow for the comparison between a control
serum sample (blue), a cortisol-deficient serum
sample (red), and an elevated Rheumatoid factor
serum sample (green; Figure 5). Each serum
sample was immunodepleted of their highabundance proteins using the Agilent Multiple
Affinity Removal column. Samples were theninjected and separated on the mRP-C18 column.
Chromatograms were overlaid and analyzed for
differences in the UV absorbance profiles. The
mRP-C18 column provides a way to fractionate at
the protein level and focus on specific fractions,with obvious differences, as shown in the inset of
Figure 5 as a potential method to identify
biomarkers.

Fractions from the regions indicated in Figure 5
were collected, dried, and resuspended in appropriate buffer. A trypsin digestion in TFE was performed on each sample. An aliquot was removed
from each sample and analyzed by LC/MS. The
LC/MS results were processed with Spectrum Millsoftware. The results for the time range between
19–21 minutes are shown in Table 4. The highlighted regions show obvious differences in proteins between the samples. The cortisol-deficientserum sample had no complement H or
apolipoprotein H, while the high rheumatoid factor
serum showed a relative loss in complement H as
noted by the lower number of spectra foundcompared to the control serum.
A similar analysis on the fraction from
29–32 minutes was also performed on the threesamples as noted by the differences in UV
absorbance (Table 5).

Expanded view
(fractions analyzed
by LC/MS)

Fractions analyzed by LC/MS

Figure 5.

6

Immunodepleted serum samples of control serum (blue), cortisol-deficient serum (red), and high rheumatoid factor
serum (green) as fractionated on the mRP-C18 column. Inset shows the expanded range of chromatograms with obvious
differences in UV absorbance. Protein loads for each sample were 300 µg.

Table 4.

Protein Identifications from Fractions of each Serum in the Time Range of 19–21 Minutes

Serum
no. spectra
total intensity
14
3.09E+07
8
1.94E+07
00.00E+00
0
0.00E+00
2
8.65E+06
1
1.84E+06
00.00E+00
0
0.00E+00

Table 5.

Def. cort.
no. spectra
total intensity
00.00E+00
0
0.00E+00
3
4.65E+06
00.00E+00
0
0.00E+00
0
0.00E+00
0
0.00E+00
0
0.00E+00

Highrheumatoid
no. spectra
total intensity
4
8.15E+06
9
2.26E+06
1
1.60E+06
2
3.54E+06
00.00E+00
2
3.13E+06
2
3.40E+06
2
4.13E+06

No. uniquepeptidesScore

Protein

12

178.37

H factor 1 (complement)

8

115

Apolipoprotein H (beta-2-glycoprotein I)

3

39.47

Ceruloplasmin

2

30.34

Complement component 1 inhibitor precursor

2

28.61

Apolipoprotein C-III precursor

2

27.34

Complement factor B preproprotein

2

24.99

Hemopexin

2

24.77

Alpha-1-acid glycoprotein 2 precursor

Protein Identifications from Fractions of each Serum in the Time Range of 29–32 Minutes

Serum
no. spectra
total intensity
37
2.04E+08
15
4.43E+07
12
7.48E+07
4
1.13E+07
4
7.05E+07
00.00E+00

Def. cort.
no. spectra
total intensity
26
1.13E+08
2
3.58E+06
1
2.30E+06
7
2.55E+07
00.00E+00
2
1.94E+06

Highrheumatoid
no. spectra
total intensity
11
2.27E+07
2
2.16E+06
11
3.84E+07
8
2.87E+07
2
2.77E+07
2
2.53E+06

1
1.56E+06

00.00E+00

0
0.00E+00

No. uniquepeptidesScore

Protein

34

555.56

Complement component 3

15

235.16

Complement component 4A preproprotein

11178.25

Apolipoprotein A-I precursor

9132.42

Antithrombin III variant

4

54.58

Apolipoprotein A-II precursor

3

36.72

Serine (or cysteine) proteinase inhibitor,clade A, member 3, precursor

1

18.22

Inter-alpha-trypsin inhibitor C-terminal

7www.agilent.com/chem
The highlighted boxes clearly show a difference inprotein levels between the control serum andabnormal sera as indicated on the chromatograms.
The high rheumatoid serum shows a lower level ofcomplement component 3 compared to the control
or cortisol-deficient serum. For both cortisoldeficient serum and high-rheumatoid-factor serum
there is a decrease in complement component 4A,
while cortisol-deficient serum has a clear decrease
in apolipoprotein A-1 protein levels. Several of
those proteins have shown a reduction in patientswith systemic lupus erythematosus (SLE), rheumatoid arthritis, and scleroderma [1]. These proteins
were easily identified by protein fractionation
after immunodepletion and reduction of sample
complexity with the mRP-C18 column while comparing UV traces of suspect serum to control
serum.

Conclusions
The Agilent mRP-C18 column provides optimized
separation for human serum after depletion of thehigh-abundance proteins using the Agilent Multiple Affinity Removal column. This combined workflow enables high recovery, excellent resolution
and reproducibility in reducing sample complexity
for biomarker research. The ability to recover >98%
with immunodepleted serum also provides theopportunity to validate biomarkers that are differentially expressed in control versus disease/treated serums. The ability to reproducibly fractionate at the protein level with high resolution
enables efficient separation and reduced overlapof proteins in collected RP fractions. Coupling of
immunodepletion with the mRP-C18 column methods allowed for rapid screening for UV absorbancedifferences from which differences in proteins
could be determined via LC/MS analysis of those
selected fractions. Researchers may be able to further optimize separation methods for use on othersamples such as membrane proteins or yeast
protein samples.

Reference
1. Grant et al., (2000) J. Immunol Methods.,
244, 41–7.

For More Information
For more information on our products andservices, visit our Web site atwww.agilent.com/chem/bioreagents.
Email technical inquiries to affinity_removal@
agilent.com
William Barrett is a Proteomics Application Scientist for Proteomic BioReagents in the Integrated
Biology Solutions Group with Agilent Technologies.
Nina Zolotarjova, James Martosella, Gordon Nicoland Hongbin Liu are R&D Scientists for Proteomic
BioReagents in the Integrated Biology Solutions
Group with Agilent Technologies.
Liang-Sheng Yang is a Manufacturing/Quality Engineer for Proteomic BioReagents in the Worldwide
Order Fulfillment Group with Agilent Technologies.
Cory Szafranski is the Product Manager forProteomics BioReagents in the Integrated Biology
Solutions Group with Agilent Technologies
Correspondance: Dr. William Barrett
Email: bill_barrett@agilent.com

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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. 2005
Printed in the USA
May 5, 2005
5989-2505EN