Discover New
Proteins Using
Immunodepletion
Proteomics
Peter Mrozinski
Application Scientist
Agilent Technologies
December 11, 2007
Introduction
Complexity and dynamic range of protein concentrations
• present a major challenge
Concentration - 10 orders of magnitude
Complexity – 106 different proteins
• can be addressed with a combination of prefractionation techniques
that deplete highly abundant proteins and fractionate
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Target Market for Multiple Affinity Removal System
The Major Application is Biomarker Discovery
Why Blood/Plasma/Serum?
ƒ Easily available
ƒ Believed to be the sample with the largest representation of the human proteome
ƒ Believed to contain subsets of the tissue proteome and xenobiotics
Other fluids such as cerebrospinal fluid, synovial fluid, urine and other body fluids can also be
used for more specific applications.
Challenges
ƒ Dynamic range of protein amounts (1010 – 1012) present
ƒ Structural complexity of these proteins.
ƒ Proteins present are not at equal concentration.
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Sample Processing Strategy
The Magic Triangle
Sample handling &
sample clean-up
Level 1
Selective filters
Multidimensional
separations
Level 2
Selective filters
Level 3
Selective filters
Identification & quantitation
by MS
Target substances
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Why the Interest in Analysis of Low Abundance
Proteins?
In human serum the concentrations of proteins are estimated to
range over twelve orders of magnitude
(mg/ml) 1
2
3
(ug/ml)
4
Level
5
(Orders of
Magnitude)
(ng/ml)
6
7
With
Immunodepletion
Level 1
Level 2
Level 3
MS
Immunoaffinity + MS
Tissue
Biomarkers
8
Still
Remaining
9
(pg/ml) 10
11
12
(fg/ml)
Number of Proteins
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Why the Interest in Analysis of Low Abundance
Proteins?
In human serum the concentrations of proteins are estimated to range over
twelve orders of magnitude
(mg/ml)
1
2
3
(ug/ml)
Level
Level 1
Level 2
Level 3
4
5
(Orders of Magnitude)
6
(ng/ml)
With Multi-Level Immunodepletion
7
8
MS
MS
Tissue
Biomarkers
Still
Remaining
9
(pg/ml)
10
11
12
(fg/ml)
Number of Proteins
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What is New in Protein Sample Preparation and Separation?
Sample Prep &
Fractionation
High Abundant
Protein Removal
Separation
Analysis
Protein
Fractionation
HPLC-Chip MS
mRP Column
Multiple Affinity
Removal System
Protein
Expression &
Purification
Protein
Characterization
Packing materials
OGE
Focus:
ƒ Recovery of sample (fewest number of steps, return of sample)
ƒ Selectivity
ƒ Reproducibility (run to run, lot to lot of product)
ƒ Reliability and increased productivity
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The Multiple Affinity Removal System
A polyclonal antibody based system to rapidly deplete multiple high abundant
proteins in serum, plasma, CSF, and other biological fluids.
H H
LH L
H L LH
LL H
H H LH
Launched in August 2003
Individual Ab Materials are mixed in
selected percentages and packed into a
column format
Apply Crude
Human Serum
Agilent continues to innovate and lead
this market
Unbound Fraction (low
abundant proteins)
Bound Fraction (high
abundant proteins)
HH
HHH
H HH
Buffer A
Buffer B
L LL
L LL L
Low-Abundant
Proteins Free from
Interferences
HH
HHH
H HH
High-Abundant
Proteins
Total Run Time (30 min)
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Importance of Orientation in Antibody Based Depletion
Antigen
Binding Site
xx
x
xx
xx
Porous Particle
xx
Fc Region
xx
Specific Antibody
xx
xx
xx
xx
xx
Versus
Crosslinker
Specific Target Protein
(eg. Albumin)
x x
x x x x x
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15.998
Representative Chromatogram of 4.6 X 100 mm Column
m AU
• Simple two buffer system
• Automatable
• Reproducible
• Low collection volumes
• HPLC and FPLC compatible
• Robust
2500
2000
5.708
1500
1000
25.933
500
0
0
5
10
15
20
25
m in
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1. Dilute and
Filter Sample
2. Remove
Buffer
3. Apply
Sample
F1
Dilute 6-8 μL human
plasma sample to 200 μL
with Buffer A. Consult
cartridge certificate for true
sample capacity. Filter
through 0.22 μm spin filter.
Remove cartridge cap and
plug and remove buffer
from top of resin bed with
transfer pipette. Never let
frits or resin bed run dry.
6. Prepare for
Elution
7. Elute Bound
Fraction
5. Wash and
Collect Flowthrough F2
4. Wash and
Collect Flowthrough F1
F1
F2
Add 200 μL diluted plasma
Add 400 μL Buffer A.
sample. Cap cartridge loosely Centrifuge 1 min at 200 x g.
or leave open. Place in 1.5
Collect in F1 tube.
mL collection tube labeled
“Flow-through fraction 1” (F1).
Centrifuge 30 sec at 200 x g.
8. ReEquilibrate
Place spin cartridge in new
collection tube labeled “Flowthrough fraction 2” (F2). Add 400
μL Buffer A. Centrifuge 1 min at
200 x g. Collect in F2 tube.
9. Analyze
F1 + F2
•Simple two buffer system
•10 minute protocol
•Reproducible
•Low collection volumes
•Only need centrifuge
•Robust
F1 + F2
Remove spin cartridge from
F2 tube and attach luer-lock
adapter tightly to top of
cartridge.
Fill 5 mL Luer-Lok™ plastic
syringe with 2 mL Buffer B and
attach to Luer-Lok adapter.
Slowly push Buffer B through
cartridge to elute bound
proteins into new collection
tube. Save eluate with
targeted high-abundance
proteins for analysis or
discard.
Fill new 5 mL plastic syringe
with 4 mL Buffer A and attach
to Luer-Lok adapter. Slowly
push Buffer A through
cartridge to re-equilibrate the
cartridge for the next sample
or store wetted with Buffer A
(at 4°C).
Re-cap both ends for storage.
Fractions F1 and F2 can be
analyzed individually or
combined. Concentrate and
analyze these fractions
containing low-abundance
proteins.
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MARS 14 Devices Offered
Format
Size
Capacity
Spin Cartridge
0.45 mL
10 uL
Column
4.6 X 50 mm
20 uL
Column
4.6 X 100 mm
40 uL
Column
7.5 X 75 mm
100 uL
Column
10 X 100 mm
250 uL
Column
Custom
Any
Bulk Media
Any
Any
Custom Blend
Any
Any
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The Agilent Multiple Affinity Removal System
¾ Selectivity
Only native human plasma proteins are used as antigens. This ensures
highest selectivity for epitopes in “real samples”. Our antibodies are so
selective that species cross-reactivity is very low.
Our buffers are specifically formulated to minimize protein-protein interactions
resulting in highest possible selectivity of binding (minimize any possible
protein-protein interactions, such as with albumin)
¾ Reproducibility
Run to run:
ƒ Coupling chemistry of antibodies to column beads is designed for
longest possible lifetime of Antibodies resulting in excellent run to run
reproducibility. Only native protein antigen is used for affinity
purification resulting in reproducible antibody selection.
ƒ Buffers for affinity purification of our polyclonal antibodies are designed
to disruption unwanted protein-protein interactions (such as with
albumin) resulting in reproducible epitope selection.
Lot to lot:
- Manufacturing processes have been engineered to provide excellent lot
to lot reproducibility
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The Agilent Multiple Affinity Removal System
¾ Ease of Use
LC column: Automated single pass, 2 buffer, 30 – 40 minute total run time to
deplete 40 - 80 uL of human plasma/serum (4.6 x 100 mm column) at 98-99%
efficiency. Larger column sizes available on request.
Spin tube: 2-step re-usable system, 10 minute total run time to deplete 10 - 15
uL of human serum/plasma
¾ Lifetime and Robustness
Antibody Binding and Buffer formulation: Antibody resin and buffers are
designed to work together – the result is unmatched lifetime and robustness
¾Compatibility with Downstream Analysis
1D gel: Proteins elute in buffer system immediately ready for application for
1DGE
HPLC: Proteins can be simultaneously concentrated, desalted, and
fractionated on our new mRP column
MS: There are no detergents present in our buffers
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Why Deplete the High Abundant Proteins?
pI 4-7
Crude serum
pI 4-7
“Removal of these proteins clearly improves the resolution in the
albumin area and increases the intensity of low abundance proteins”
Depleted serum
Courtesy of Dr. Tasso Miliotis, Karin Björnhall and Dr. Pia Davidsson, Experimental Medicine/Molecular Sciences, Astra Zeneca, Mölndal, SE
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Why Multiple Affinity Removal System?
α
γ β
Plasma
Plasma after Depletion
Data: Dr. Y.K. Paik
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Agilent Multiple Affinity Removal System:
Where Are We? & What is Next?
2004
“Original” Top-6
Human Serum
Spin Tube format
2006
2005
“High Capacity” Top-6
Human Serum
“High Capacity” Top-7
Human Plasma
2007
Level-II
Human-14
Spin Tube format
While Maintaining our Focus:
Mouse-3
ƒ Recovery of sample (fewest number of steps, return of sample)
ƒ Selectivity
ƒ Reproducibility (run to run, lot to lot of product)
ƒ Reliability and increased productivity
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MARS Human 14
Anti – HSA
Anti – IgG
Anti – Transferrin
Anti – Haptoglobin
Anti – Alpha1-antitrypsin
Anti – IgA
Anti – Fibrinogen
Anti – Alpha2-Macroglobulin
Anti – Alpha1-Acid Glycoprotein
Anti – IgM
Anti – Apolipoprotein AI
Anti – Apolipoprotein AII
Anti – Complement C3
Anti – Transthyretin
Proteomic Sample
High Abundance Protein
Low Abundance Protein
Proteomic Sample with Low Abundance Proteins
In Column Flow Through
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MARS Human 14
L L
L L L
L L
H H
L H L
H L LH
L H
H LH L H
IgG
Transferrin
Fibrinogen
IgA
Haptoglobin
α1-Antitrypsin
15%
Complement C3
α1-Acid
Glycoprotein
Apolipoprotein AII
Transthyretin
Apolipoprotein AI
6% Remaining
for Analysis
IgM
Albumin
α2-Macroglobulin
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Overlay of Chromatograms From Run 1, 50, 100, 150 and 200
on a Human 14 Column
No1rm .
4.6 mm ID x 100 mm column
Bound Fraction
2500
2000
Column performs
reproducibly for 200+ runs
Flow-through
Fraction
1500
Plasma
Injection
1000
Elution
0.125 ml/min
Re-equilibration
1 ml/min
500
0
0
Figure 3
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5
10
15
20
25
30
35
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m in
SDS-PAGE Analysis of Human14 Reproducibility
Runs
kDa
200.0
116.3
97.4
66.3
55.4
36.5
31.0
21.5
14.4
6.0
1, 50, 100, 150, 200
123456-
Mark12 Standards
Flow-through from Run 1
Flow-through from Run 50
Flow-through from Run 100
Flow-through from Run 150
Flow-through from Run 200
The correct binding and elution
formulations are required for
reproducible long life use.
Reproducible depletion of target
proteins from human serum as
indicated by constant gel pattern of the
depleted serum.
Protein content of flow-through
fractions remains consistent during 200
runs.
SDS-PAGE Analysis of Fractions from MARS14 Column
1
kDa
200
116
97
66
55
37
31
22
14
6
2
3 4
Serum
5
6
7 8
Plasma
9
1 – MultiMark Standards
2 - Serum
3 - Serum Flow-through Fraction
4 - Serum Bound Fraction
5 - MultiMark Standards
6 - Plasma
7 - Plasma Flow-through fraction
8 - Plasma bound fraction
9 - MultiMark Standards
* For 4.6x100 mm column – 40μl sample loading
results in the 94% total protein depletion from serum
(~194µg of protein in the flow-through fraction) and
92% of total protein depletion from plasma (~270μg of
protein in the flow-through fraction).
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ELISA Results for the Depletion Efficiency After 200 Runs *
Target Protein
Depletion in Serum
Depletion in Plasma
Albumin
99.9%
99.6%
Haptoglobin
99.0%
98.9%
Transferrin
98.6%
99.5%
IgG
99.6%
99.5%
IgA
99.2%
99.5%
α1- Anti-trypsin
98.0%
99.5%
α2-Macroglobulin
99.6%
99.3%
α1-Acid Glycoprotein
99.6%
99.5%
Apolipoprotein AI
99.2%
98.4%
Apolipoprotein AII
96.0%
95.0%
Complement C3
96.0%
98.6%
IgM
99.3%
99.5%
Transthyretin
99.3%
97.5%
Fibrinogen
N/A
97.6%
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Selectivity of Human-14 Column
Proteins identified in the serum bound fraction by LC/MS/MS
1
Human Serum Albumin
2
Immunoglobulin G
3
Immunoglobulin M
4
Immunoglobulin A
5
Haptoglobin
6
Transferrin
7
Alpha1-Anti-trypsin
8
Alpha2-Macroglobulin
9
Complement C3
10
Alpha1-Acid Glycoprotein
11
Apolipoprotein AI
12
Transthyretin (prealbumin)
13
Apolipoprotein AII
14*
Apolipoprotein B-100
15*
Plasma protease C1 inhibitor
16*
Zinc-alpha-2-glycoprotein
17*
Apolipoprotein L1
* Untargeted proteins not quantitatively removed
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1D
Proposed Workflow:
Removal of the 14 most abundant proteins
with the Agilent Human-14 column
mRP Column
Immunodepletion
Simultaneously:
ƒ Concentrate
ƒ Desalt
ƒ Fractionate
Low Abundance proteins
High Abundance
proteins
2D
2D
mRP Column
HPLC-Chip /
XCT Ultra Trap
Digest
1.
2.
3.
4.
Depletion
Protein Fractionation
Peptide Fractionation
Peptide Separation
Robust integrated
nanospray system
3D
+
4D
32 mRP fractions analyzed
after tryptic digestion 14 salt steps per fraction
Spectrum Mill processed the
resulting 448 data files as
one data set
HPLC-Chip:
Enrichment column
RP Separation Column
10-port valve
Nano-electrospray tip
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Human Plasma
Immunodepletion of 14
high-abundant proteins
on Human-14 column
Sample Denaturation
Reversed-phase separation of the
depleted plasma on mRP-C18 column
TFE-based tryptic digestion of RP fractions
Peptide analysis by 2D HPLC-Chip LC/MS/MS system
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Partial List of Low Level Protein Identified from Plasma
Number
Protein Name
Accession #
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Insulin-like growth factor binding protein 3 (ng/mL)
Transitional endoplasmic reticulum ATPase (TER ATPase)
Intercellular adhesion molecule-2 precursor (ICAM-2) (ng/mL)
Mitogen-activated protein kinase 3 (MAPK)
Polycystic kidney and hepatic disease 1 precursor
Dopamine beta-monooxygenase precursor
ADAM 10 precursor (ng/mL)
Integrin alpha-1 (CD49a)
Peripheral-type benzodiazepine receptor-associated protein 1 (PRAX-1)
Insulin-like growth factor binding protein 4 precursor (IGFBP-4) (ng/mL)
6-phosphofructokinase, liver type
cGMP-dependent protein kinase 1, beta isozyme
Cadherin-13 precursor (ng/mL)
Serine/threonine-protein kinase MAK
Insulin-like growth factor binding protein 6 precursor (IGFBP-6) (ng/mL)
Tyrosine-protein kinase JAK1
Aminopeptidase N
Tumor necrosis factor, alpha-induced protein 2 (pg/mL)
Protein kinase C, mu type (nPKC-mu)
Interleukin-1 receptor-like 1 precursor (pg/mL)
Cyclin T2
Interleukin-27 beta chain precursor (IL-27B) (pg/mL)
LIM domain kinase 1 (LIMK-1)
Protein-tyrosine phosphatase precursor
P17936
P55072
P13598
Q8IVH8
Q8TCZ9
P09172
O14672
P56199
O95153
P22692
P17858
P14619
P55290
P20794
P24592
P23458
P15144
Q03169
Q15139
Q01638
O60583
Q14213
P53667
Q12913
Distinct
Peptide #
6
7
4
6
5
3
4
4
4
2
4
3
3
3
2
3
2
2
2
2
2
2
3
2
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Outlook
• Human-14 Multiple Affinity Removal Column depletes the top 14 abundant
proteins from human serum, plasma, CSF, and other biological fluids.
• The Multiple Affinity Removal System provides ~94% total protein mass
removal from plasma/serum and longest column lifetime when compared
to other products. This translates to the lowest cost per ml of depleted
sample.
• The Multiple Affinity Removal System also provides the greatest ease of
use, highest selectivity and reproducibility (run to run, lot to lot) compared
to any equivalent product.
• Protein prefractionation by immunodepletion, mRP and OGE enables one
to dive deeper into the plasma proteome and provides methods compatible
with LC-MS based analysis.
• All prefractionation methods and tools integrate well together minimizing
sample loss due to excessive sample manipulations
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