New Sample Preparation and Protein Fractionation Techniques
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
What is New in Protein Sample Preparation and Separation? Sample Prep & Fractionation
High Abundant Protein Removal
Separation
Protein Fractionation mRP Column
Multiple Affinity Removal System
Analysis
Protein Expression & Purification
Protein Characterization
HPLC-Chip MS Packing materials
OGE
Focus:
Recovery of sample (fewest number of steps, return of sample) Selectivity Reproducibility (run to run, lot to lot of product) Reliability and increased productivity
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
The Multiple Affinity Removal System A polyclonal antibody based system to rapidly deplete multiple high abundant proteins in serum/plasma/CSF.
H H L HL L HL H 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) Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
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: 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. 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
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
The Agilent Multiple Affinity Removal System ¾ Ease of Use LC column: Automated single pass, 2 buffer, 30 minute total run time to deplete 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 15 uL of human serum/plasma
¾ Compatibility 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
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Why Multiple Affinity Removal System?
D J E
Plasma
Plasma after Top-6 Depletion
Data: Dr. Y.K. Paik Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Agilent Multiple Affinity Removal System: Where Are We? & What is Next? FY2004
FY2005
FY2006 (April 2006)
“Original” Top-6 Human Serum
“High Capacity” Top-6 Human Serum
“High Capacity” Top-7 Human Plasma
+ Spin Tube format
Spin Tube format
“High Capacity” Level-II human
While Maintaining our Focus: Mouse-3
Recovery of sample (fewest number of steps, return of sample) Selectivity Reproducibility (run to run, lot to lot of product) Reliability and increased productivity
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Selectivity of Plasma-7 Column Proteins Identified in Bound Fraction by LC/MS/MS Flow Through Fractions from different amounts of loaded plasma sample Human Plasma STD
Bound Fraction 60 uL 70 uL 80 uL
Untargeted Proteins
Gel Band # 1 2 3 4 5 6 7 8 9 10 11
kDa 200.0 116.3 97.4 66.3 55.4
36.5
12 13 14 15 16
31.0 21.5 14.4 6.0
4-20% 420% SDS -PAGE
Proteins Identified
Fibrinogen Fibrinogen, Haptoglobin , HSA IgG, IgA, IgA Complement C3 IgG, HSA, IgA IgG, HSA IgG, HSA, Ceruloplasmin HSA, Haptoglobin, IgG HSA, Complement B pre IgG, HSA Transferrin, HSA HSA, Transferrin, Alpha-1-antichymotrypsin Alpha1 -Antitrypsin, HSA HSA Apolipoprotein H Apolipoprotein Hemoglobin alpha chain
ELISA analysis indicate 99.4% depletion of Fibrinogen from 60, 70 and 80 μl of a plasma load on a 4.6x100mm column
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Reproducibility from Run to Run Comparison of runs 40, 80, 120, 160, & 200 Std 1
SDS-PAGE analysis of the flowthrough fractions from multiple runs on a Human Plasma 7 column
1
2
40 3
80 120 160 200 4
5
6
78
kDa 200.0 116.3 97.4 66.3 55.4
Bound Fraction (Bands excised for confirmation of ID by MS/MS)
36.5 31.0
10 μg of protein/well
21.5 14.4 6.0
4-20% SDS-PAGE
Flow-through Fraction
1- Human Plasma 2- Mark12 Standards 3- Flow-through Fraction, Run 1 4- Flow-through Fraction, Run 40 5- Flow-through Fraction, Run 80 6- Flow-through Fraction, Run 120 7- Flow-through Fraction, Run 160 8- Flow-through Fraction, Run 200
Column performs reproducibly for 200 runs!
Bound Fraction
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
mRP-C18 High-Recovery Protein Fractionation Column
mRP (macroporous reverse-phase)
What is it?
Reverse Phase column for protein separation and fractionation. The silica based particles and recommended LC methods have been optimized for: Highest recoveries of protein samples (95% - 99% of loaded sample) Highest resolution separations Reproducibility High sample loading capacity (3X higher than most standard RP columns) Lifetime
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Comparison of mRP with Zorbax SB300-C8
Norm.
SB300-C8 40
hemopexin
30
Absorbance (280 nm)
Absorbance (280 nm)
mRP-C18
mAU
30
20
A1 Apolipoprotein
Acid-glycoprotein 25 20
complement component C4 15 10
10
5 0 0 0
10
20
30
Retention Time
40
50 min
0
10
20
30
40
50
min
Retention Time
Sample: 270ug flow-through (6M urea/5.0% AcOH) of immunodepleted human serum from Multiple Affinity Removal System column Sample: 270ug flow-through (6M urea/5.0% AcOH) of immunodepleted human serum from Multiple Affinity Removal System column Columns: Panel A – Zorbax SB300-C18 (300 A, 5.0um), 4.6 mm x 50 mm i.d., SS; Panel B – mRP-C18 (macroporous, 5um), 4.6 mm Columns: Panel A – Zorbax SB300-C18 (300 A, 5.0um), 4.6 mm x 50 mm i.d., SS; Panel B – mRP-C18 (macroporous, 5um), 4.6 mm x 50 mm i.d., PEEK, 0.75mL/min., DAD 280nm x 50 mm i.d., PEEK, 0.75mL/min., DAD 280nm Mobile Phase & Conditions: A-0.1% TFA/water, B-0.08%TFA/ACN, Temp 80° C, gradient:5-30%B in 5min., 30-55%B in 33min., 55-100%B in 4min. Mobile Phase & Conditions: A-0.1% TFA/water, B-0.08%TFA/ACN, Temp 80° C, gradient:5-30%B in 5min., 30-55%B in 33min., 55-100%B in 4min. 1D SDS PAGE: Collected 36 fractions (1.0 min. time slices) from immunodepleted human serum RP separation 1D SDS PAGE: Collected 36 fractions (1.0 min. time slices) from immunodepleted human serum RP separation
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Protein Fractionation on mRP (4.6 x 50mm mRP-C18) mAU
mAU
7
8
6
7
5
6 5
4
4 3
3 2
2
1
1
00
10
20
30
40
50
60
70
80
00
min
Hela Membrane Prep
10
20
30
40
50
HeLa cell lysate (352ug)
Highest Recovery
Lipids
mAU mAU
25
25
20
20
15
15
10
10
5
5
0
0
60
10
20
30
40
“Top-6” depleted human serum
50
min
0 0
10
20
30
40
50
60
min
Human Brain membrane lipid Raft prep (500ug)
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Preliminary LC-MS data of the lipid fraction isolated from the human brain membrane rafts sample LIPID ELUTION 760.5 m/z C34:1 PC 600000 PC = phosphatidylcholine SM = sphingomyelin
500000
400000
734.5 m/z C32:0 PC
300000
200000
731.5 m/z C18:0 SM 788.5 m/z C36:1 PC 732.5 m/z C32:1 PC
813.6 m/z C24:1 SM
100000 703.5 m/z C16:0 SM 0 5 0
10 10
15 20
20 30
25
min 40
50
60
min
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Lipid Raft Sample: mRP Fractionation followed by 1D-SDS PAGE Fractionation 5 11
7
4
1
12
6
2
10
8 9
13
3
Selected Excised Bands Which are Intregal Membrane Proteins 1. 2. 3. 4. 5. 6. 7.
Voltage-Dependent Anion Selective Channel Protein 1 Cytochrome C Oxidase subunit IV (COX IV) Cytochrome C Oxidase subunit IV (COX IV) 2’,3’-Cyclic-Nucleotide 3”-Phosphodiesterase (CNP) Spectrin Alpha Chain, Brain (Alpha-II Spectrin) Vacuolar ATP Synthase Subunit E Creatine Kinase, B Chain
8. 9. 10. 11. 12. 13. 14.
ATP Synthase alpha chain Vacuolar ATP Synthase Subunit D Vacuolar ATP Synthase Subunit B Contactin Associated Protein Vacuolar ATP Synthase Subunit C ATP Synthase Chain B Thy-1 Membrane Glycoprotein Precursor (Thy1)
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Lipid Raft Sample: Reproducibility and Baseline Stability
Absorbance (280 nm)
Overlay of 5 Chromatograms (Lipid Raft Sample)
Time (min.)
Absorbance (280 nm)
Baseline Before and After Sample Injection
Blue – postrun column injection after 5 separations
Red – prerun column injection
Time (min.)
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Hela Cell Membrane mRP Fractions followed by 1D-SDS 4.6 x 50mm mRP PAGE Fractionation Hela membranes, starting material, 22 μg 1 2 3 4 5 6 7 Gel 1
8
9
10
1
2
3
4
5
6
7
8
9 10
Gel 2
kDa 200.0 116.3 97.4 66.3 55.4
Lipids
36.5 31.0 21.5 14.4 6.0
Stds
Stds
mRP Fractions
mRP Fractions
Identification performed by chip-based nano LC/MS/MS from excised gel bands Sample (216 gel bands from mRP)
HeLa Membranes
Total Acquisition Time (hrs)
108
# MS/MS Spectra Collected
486,700
Peptides # Distinct Matched Peptides Matched
3841
# Total Proteins Identified
688
# Membrane Proteins Identified
364
# Integral Membrane Proteins Identified
286
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Hela Cell Lysate mRP Fractionation followed by 1D-SDS PAGE Fractionation 4.6 x 50mm mRP C18 column kDa
M
1
2
3
4
5
6
7
8
9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25 26
200.0 116.3 97.4 66.3 55.4 36.5 31.0 21.5 14.4 6.0
mRP Column
mAU 8 7 6 5 4 3 2 1 00
10
20
30
40
50
60
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
For Plasma/Serum Biomarker Discovery: Combine “Top-6” and mRP Fractionation Removal of 6 most abundant proteins
Fractionation of lower abundance proteins
MARS Immunodepletion Low abundance proteins
Two fractions analyzed by MS tryptic digestion
LC/MSD Trap XCT
mRP Column Fractionation Simultaneously: Concentrate Desalt Fractionate
Blue
Control Serum
Red
Cortisol-deficient Serum
Green Rheumatoid Serum
Spectrum Mill High serum # def. Cort.Rheumatoid spectra # spectra # spectra total total total # Unique intensity intensity intensity Peptides Score Protein 14 0 4 3.09E+070.00E+00 8.15E+06 12 178.37 H factor 1 (complement) 8 0 9 1.94E+070.00E+00 2.26E+07 8 115 apolipoprotein H (beta-2-glycoprotein I) 0 3 1 0.00E+004.65E+06 1.60E+06 3 39.47 ceruloplasmin 0 0 2 0.00E+000.00E+00 3.54E+06 2 30.34 complement component 1 inhibitor precursor 2 0 0 8.65E+060.00E+00 0.00E+00 2 28.61 apolipoprotein C-III precursor 1 0 2 1.84E+060.00E+00 3.13E+06 2 27.34 complement factor B preproprotein 0 0 2 0.00E+000.00E+00 3.40E+06 2 24.99 hemopexin 0 0 2 0.00E+000.00E+00 4.13E+06 2 24.77 alpha-1-acid glycoprotein 2 precursor
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Off-Gel Electrophoresis Technology and Applications
For Research Use Only. Not for use in diagnostic procedures.
Strategy Towards Low Abundance Proteins with Immunodepletion & OGE
Fraction I MARS Separation
OGE Fractionation
Removal of known proteins or protein classes (specific)
Fractionation and enrichment of unknown proteins and peptides (generic)
Î reducing dynamic range
Î reducing sample complexity
Crude sample
High-abundance protein-free fraction
Fraction II
Enriched, low-abundance protein fractions
MARS: Multiple Affinity Removal System OGE: Off-Gel Electrophoresis Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
pI-based Fractionation: Off-Gel-Electrophoresis
pH gradient
immobilized (IPG gels)
Number of fractions
d 30
Fraction volume
0.05 - 0.1 ml
Resolution
0.05-0.5 pH
Separation time
5 - 20 h
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Plasma Proteome Workflow Immunodepletion, OGE, LC/MS
H LHLHL H LHLLHHLH H
Multiple Affinity Removal Column
Off-Gel Electrophoresis
Workflow and instrumental setup of the immunoaffinity column, OGE and LC/MS system
HHHH HH HH
LLL LLLL
HHH H HH HH LC/MS system Solvent tray Orthogonal nanospray source and nanocolumn
Degasser
nanoflow pump
P-WPS
6-port micro switching Valve and holder
Solvent tray 2nd pump
Cooler
LC/MSD Trap XCT
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Analysis of OGE Fractions by 1D Electrophoresis fraction #
4
*
1/2 3
4
5
6
7
8
9 10 11 12 13 14 15
E. coli cell extract Coomassie Brillant Blue stain * unfractionated sample
5
pH
6
7
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Analysis of OGE Fractions by High Resolution 2DE Albumin-Depleted Human Plasma, Silver Stain; Experiment done by Lab of Prof. Tissot/CHUV target: 'pH 0.16 pH 4.5 – 5.5
pH 4.5 – 5.5
pH 4.5 – 5.5
pH 4.5 – 5.5
fraction #4
fraction #5
fraction #6
fraction #7
pH 4.77-4.93
pH 4.93-5.10
pH 5.10-5.26
pH 5.26-5.42
almost no overlap between fractions Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Applications Protein Samples: • Immunodepleted serum/plasma • Cerebrospinal fluid (CSF) • HeLa proteasome cell extract • Mammalian macrophage cell extract • preB 697 cell extract • Bacterial lysates • E. coli • H. influenzae
Peptide samples
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
OGE Fractionation of a Protein Fraction from HeLa S3 Cell Extract* analyzed by Chip-LC/MS
Proteins sorted by increasing calculated pI
Fraction 1-15, pH 3-10
Prefractionation of this cell extract leads to significantly greater number of proteins identified: 144 proteins total for 15 OGE fractions 20 proteins total for 15 injections of unfractionated sample
* binding to anion exchange resin Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
OGE Fractionation of Peptides - E.Coli Tryptic Digest Average Peptide pI with Standard Deviation for Autovalidated Peptides Auto validated peptides 10.0 9.0
calc pI
8.0 7.0 6.0
pH in center Average peptide_pI
5.0 4.0 3.0 0
5
10
15
20
25
Fraction
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
OGE Fractionation of Peptides - E.Coli Tryptic Digest Number of Peptide Identifications in Number of Fractions Bar Chart
64,6%
670 proteins identified with Autovalidation criteria
2265 2250
2000
3454 distinct peptides identified
1750
=> The majority of peptides are found in only 1 or 2 fractions!
5840 spectra
1500
1250
1000
19.2% 750
674
500
7.1% 251 2.9%
250
102 0
1
2
3
4
43
20
17
6
4
3
1
1
1
5
6
7
8
9
10
11
13
16
F25
Fractions Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
OGE Fractionation of Peptides Trypsin Digested E-Coli Lysate Peptide scores plotted against delta pI (3454 peptides, S catter P lotafter (2) autovalidation) 5840 spectra, valid
Peptide scores plotted against delta pI (5251 spectra, not valid after autovalidation) S catter P lot (2)
25
25
20
20
15
15
10
10
5
5
-8
-6
-4
-2
delta pI
0
2
4
6
-8
-6
-4
-2
0
2
4
6
delta pI
Can be used to search for charged PTM’s, peptides with high delta pI and high score are possible candidates. Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
OGE Fractionation of Peptides Trypsin Digested E-Coli Lysate Peptide scores plotted against delta pI (11091 spectra, S catter P = lot green, (2) valid after autovalidation not validated = red)
Peptide scores plotted against delta pI (1964 not S catter P (2) pH of center pH of well) validated spectra within ±lot0.63
25
25
20
20
15
15
10
10
5
5
-8
-6
-4
-2
delta pI
0
2
4
6
-8
-6
-4
-2
0
2
4
6
delta pI
~35% of spectra not autovalidated are within ± 0.5pH. Using this information, up to 20% more peptides can be identified! Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Pseudo 2D Gel: OGE Fractions of Human Serum analyzed with the Protein 200-HT2 Assay on Agilent’s 5100 ALP fraction pH:
4.21 4.32
4.43 4.54
4.66 4.77
4.88 4.99
5.11 5.22 5.33
5.44 5.56 5.67
5.78 5.89
6.01 6.12 6.23
6.34
6.46 6.57 6.68
6.79
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Outlook • Protein prefractionation by immunodepletion, mRP and OGE enables a deeper dive 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 • OGE provides PTM-grade resolution of proteins and peptides and delivers fractions in solution (LC/MS compatibility) • The mRP column provides highest recovery of protein samples (95+%) even on challenging protein samples such as integral membrane proteins • The Multiple Affinity Removal System provides the highest sample capacity per ml resin 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.
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
Further Information Bioreagents catalog – 5989-3431EN "The 2005-2006 Bioreagents Product and Resource Guide" CD Compendium – Bioseparations 5989-4047EN Weblink: http://www.agilent.com/chem
Pub 5989-5136EN For Research Use Only. Not for use in diagnostic procedures.
