Performance Evaluation of Novel Affinity Ligands for Immunodepletion
Majlinda Kullolli, Jonathan Warren and Sharon J. Pitteri
Department of Radiology, Stanford University
Flow-through fractions were digested with trypsin and analyzed by LC-MS/MS to investigate the protein
capture efficiency and the column capacity as shown in Figure 3.
125
Spectral Counts Identified
Human plasma is one of the most commonly used diagnostic fluids in clinical chemistry. Understanding the
plasma proteome would facilitate the search for disease biomarkers. The plasma proteome is a challenge to
modern analytical technologies due to the large dynamic range of protein abundance. The 22 most abundant
plasma proteins represent approximately 99% of the total protein mass in plasma. Reducing the concentration
of these abundant proteins in plasma is a commonly used strategy to aid the analysis of medium to low
abundant proteins. Affinity-based pre-fractionation of proteins coupled to LC-MS/MS has been shown to be
an effective means of reducing the dynamic range of protein abundance. Several multicomponent affinity
matrices based on antibody capture to target the most abundant plasma proteins are commercially available.
These antibody columns are efficient and reproducible for removal of the targeted proteins.
Here we evaluate a novel class of affinity ligands derived from Camelid heavy chain antibody produced by
Saccharomyces cervisiae (CaptureSelect®). These ligands provide high-affinity, high-capacity, highspecificity binding, and are readily manufactured in an animal-free system offering an inexpensive alternative
to traditional antibody-based matrices. These ligands provide a flexible platform for immundepletion as their
use can be customized depending on the amount of plasma to be depleted. 1D gel-electrophoresis and LCMS/MS methods were used to examine the following figures of merit: efficiency of depleting the top 14 most
abundant proteins, column binding capacity, linearity, reproducibility, and non-specific binding.
IGA
IGM
100
IgG-4
ApoA1
IgKC
IgD
75
50
25
20
0
17
5
15
0
12
5
10
0
75
0
50
0
25
Plasma Volume Loaded
(uL)
Methods
Figure 7. Correlation of two independent depletion experiments.
Removal of Additional Abundant Proteins
Figure 3. LC-MS/MS analysis of the flow-through fraction.
Linearity of CaptureSelect® Human 14 Depletion Column
To study the linearity of the column, five plasma injections were made onto the column as shown in
Figure 4. A high linear correlation was observed for the flow-through and the bound fraction with R2 of
0.921 and 0.9951, respectively.
350
300
R² = 0.921
250
200
After the removal of the top 14 most abundant proteins, the next most abundant protein is complement
C3, which was identified with up to 1232 total peptides in the flow-through fraction. To further
decrease the dynamic range of plasma, we depleted complement C3, C4 and C1q. As shown in Figure
8a, plasma was first depleted of the 14 most abundant proteins. The depleted plasma was then loaded
onto a 3 mL gravity column packed with C3, C4, and C1q ligands. The column was incubated for 30
min, the flow-through was washed with PBS, and the bound proteins were eluted with 100 mM
glycine, pH 2.5. The flow-through and bound fractions were concentrated to 100 uL and analyzed by
1D SDS-PAGE (Figure 8b) and LC-MS/MS. The resulting number of the peptides for complement C3
decreased by 95%, thus enhancing the identification of low abundant proteins such as annexin A2,
protein S-100A8, and protein S-100A9.
Plasma
( 125 uL)
150
0
1. Binding Capacity and Linearity of the CaptureSelect® Human 14 Depletion Column
15000
2. Reproducibility of the CaptureSelect® Human 14 Depletion Column
5000
Bound
0
50
100
150
200
250
195
125
125
Bound
%
Recovery
%
Mean
7.09
93.11
100.2
Std Dev
0.66
2.98
2.62
CV
9.32
3.20
2.62
Flow-through
Table 1. Five replicate runs were
performed on the depletion column to
evaluate column reproducibility and recovery.
MW kDa
FT-Run 5
FT-Run 4
FT-Run 3
FT- Run 2
FT- Run 1
Plasma Load
The reproducibility of the depletion column was evaluated by 1D SDS-PAGE and stained using
Coomassie Blue. As seen in Figure 6, the protein patterns observed across replicates is reproducible for
the flow-through (Figure 6a) and the bound fraction (Figure 6b)
195
195
125
80
80
80
50
40
50
50
40
40
Bound 50 uL-2
40
25
Complement C3, C4 and C1q
Column
20
b
Flow Through (FT)
(96 uG or 32 %)
Bound
(135 uG or 45%)
a
Figure 8. a) The tandem immunodepletion of plasma workflow, b) 1D SDS-PAGE analysis of the complement
C3, C4, and C1q depleted fractions.
Here, we investigated the performance of novel affinity ligands to deplete 17 abundant proteins in
plasma (albumin, IgG, IgM, IgA, IgE, IgD and free light chains, transferrin, fibrinogen, α-1
antitrypsin, α-2 macroglobulin, α-1 acid glycoprotein, apolipoprotein A1, haptoglobulin, complement
C3, C4, and C1q) allowing effective binding of high abundant proteins in a very reproducible manner.
The depletion of 17 abundant proteins improves the identification of low abundant proteins which is
crucial for biomarker discovery. An advantage of these ligands is that the material can be packed for
any size column depending on the proteomics platform. Furthermore, the plasma is in the native
condition which makes it deal for sample preparation prior to other coupled analytical separation
methods such as strong cation exchange, reversed-phase, 1D SDS-PAGE, LC-MS/MS, and multiple
reaction monitoring for biomarker discovery validation. Additional merits of the CaptureSelect®
material include that the material can be re-used for up to 100 runs, and the inexpensive nature of the
material makes it suitable for single-use spin column applications.
25
25
25
20
Bound 50 uL-1
Bound 25 uL-2
Bound 25 uL-1
50
Acknowledgements
50
40
25
Bound-14P
(5400 uG or 93%)
Figure 5. Chromatographic profile the depletion
125
80
Flow Through-14P (FT)
(300 uG or 7%)
Conclusions
Time (min)
MW kDa
Bound 250 uL
Bound 200 uL
Bound 150 uL
Bound 125 uL
Bound 100 uL
Bound 75 uL
Bound 50 uL
Bound 25 uL
Plasma Load
MW kDa
FT 250 uL
FT 200 uL
FT 150 uL
FT 125 uL
FT 100 uL
FT 75 uL
FT 50 uL
FT 25 uL
195
n=5
Flow-Through
%
Bound -Run 5
250
To further investigate the binding capacity of the depletion column we performed 1D SDS-PAGE on the
flow-through and bound fractions for all 8 different amounts of plasma loaded onto the column (25-250 uL)
shown in Figure 2 a and b, respectively. As seen in the Figure 2a, the column starts saturating at 125 uL. The
specificity of the immunodepletion column is a critical parameter. To determine the unspecific binding of
proteins, we analyzed the bound fraction on a 1D SDS-PAGE shown in Figure 2b and by LC-MS/MS.
Plasma Load
80
To validate the reproducibility of the column, repetitive runs were performed (n=5). Protein
concentration in each fraction was measured by Bradford assay as shown in Table 1. An average of 93%
of proteins in plasma bound to the depletion column in a reproducible manner, with good protein
recoveries (100%), and coefficient of variations (CV) of approximately 9.3% and 3.2% for the flowthrough and bound fractions, respectively
a
b
Plasma Volume Loaded
Plasma Volume Loaded
(uL)
(uL)
Figure 1. Loading capacity of the depletion column, a) showing the amount of proteins in the flow through, b) the
amount of the proteins bound onto the column
MW kDa
125
Bound -Run 4
200
195
Figure 4. Column linearity for the flow-through (a) and bound fraction (b)
Bound - Run 3
150
b
Bound -Run 2
100
a
15
0
50
125
10000
0
0
100
Bound -Run 1
400
75
Amount of Plasma Loaded (uL)
Intensity (mVolts)
800
50
Plasma Load
1200
Amount of Proteins
Depleted (ug)
Amount of Proteins in the
Flowthrough (ug)
The characterization of the depletion column was performed using pooled reference plasma. Column
capacity was studied by injecting 25, 50, 75, 100, 125, 150, 200, and 250 µL of crude plasma. Figure 1
shows the amount of protein in the flow-through (a) and bound (b) to the depletion column measured by
Bradford assay. As seen from the figure, at 125 uL of crude plasma the column starts showing saturation.
Thus, using a 5.6 mL depletion column 125 uL of plasma can be depleted.
25
FT 50 uL-2
14 Protein Depletion Column
0
FT 50 uL-1
50
FT 25 uL-2
Results
FT 25 uL-1
100
Plasma Load
Amount of the Protein
in the Flow Through (uG)
CaptureSelect® Human 14 packing material (removes albumin, IgG, IgM, IgA, IgE, IgD and free light
chains, transferrin, fibrinogen, α-1 antitrypsin, α-2 macroglobulin, α-1 acid glycoprotein, apolipoprotein A1,
and haptoglobin) was packed in a 5.6 mL omni glass column. Column characteristics were evaluated using
the following method. Prior to depletion of abundant proteins, plasma was diluted 1:5 with PBS. Sample was
loaded onto the column at a flow rate of 0.5mL/min. The flow-through was washed with PBS and then
collected and concentrated to 100 uL using the 3kDa cutoff Amicon filters. Bound proteins were eluted with
100 mM glycine, pH 2.5. The depletion column was then equilibrated with 10 column volumes of PBS. Total
protein concentration was measured by Bradford assay. To validate the column performance, the flowthrough fraction and bound fraction were subjected to 1D SDS-PAGE and LC-MS/MS.
Data Analysis. LC-MS data were searched using the Computational Protein Analysis System using
X!Tandem (human UniProtKB database). Protein identifications required at least two peptides with
PeptideProphet score >0.75.
MW kDa
Overview
To evaluate the reproducibility of the depletion column, two replicate flow-through fractions were
digested with trypsin and analyzed by LC-MS/MS. Figure 7 shows the correlation of the two
independent runs (R2=0.966).
Developmental Cancer Research Award, Stanford Cancer Institute 2011-2012
20
20
20
Canary Foundation
a
Figure 2. Binding capacity of the depletion column via SDS-PAGE a) flow-through of the depletion column b) the
bound fraction of the depletion column
b
a
Figure 6. Replicate runs analyzed by 1D SDS-PAGE for the flow-though (a) and the bound fraction (b)
b
BAC CaptureSelect