New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research...
Transcript of New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research...
Pub 5989-5136EN
New Sample Preparation and Protein Fractionation
Techniques
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
What is New in Protein Sample Preparation and Separation?
ProteinExpression & Purification
ProteinCharacterizationSeparationSample Prep &
FractionationAnalysis
High Abundant Protein Removal
Protein Fractionation
HPLC-Chip MS
Packing materialsmRP Column
Multiple Affinity Removal System OGE
Focus:Recovery of sample (fewest number of steps, return of sample)SelectivityReproducibility (run to run, lot to lot of product)Reliability and increased productivity
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
The Multiple Affinity Removal SystemA polyclonal antibody based system to rapidly deplete multiple high abundant proteins in serum/plasma/CSF.
Launched in August 2003Individual Ab Materials are mixed in selected
percentages and packed into a column formatAgilent continues to innovate and lead this
market
Low-Abundant Proteins Free from Interferences
Apply Crude Human Serum
High-Abundant Proteins
LL LL LL L
HH HHHHHH
H
H
LLLH
H
HH HHH LL
LL
HHHHHHHH
Buffer B
Buffer A
Bound Fraction (high abundant proteins)
Unbound Fraction (low abundant proteins)
Total Run Time (30 min)
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
The Agilent Multiple Affinity Removal SystemSelectivity
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)
ReproducibilityRun 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 designedto disruption unwanted protein-protein interactions (such as with albumin) resulting in reproducible epitope selection.
Lot to lot: Manufacturing processes have been engineered to provide excellentlot to lot reproducibility
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The Agilent Multiple Affinity Removal System
Ease of UseLC 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 ofhuman serum/plasma
Compatibility with Downstream Analysis1D gel: Proteins elute in buffer system immediately ready for application for 1DGEHPLC: Proteins can be simultaneously concentrated, desalted, and fractionated on our new mRP columnMS: There are no detergents present in our buffers
For Research Use Only. Not for use in diagnostic procedures.
Why Multiple Affinity Removal System?
Plasma Plasma after Top-6 Depletion
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Data: Dr. Y.K. Paik
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Pub 5989-5136EN
Agilent Multiple Affinity Removal System: Where Are We? & What is Next?
“Original” Top-6 Human Serum
Spin Tube format
Mouse-3
“High Capacity” Top-6 Human Serum
Spin Tube format “High Capacity”Level-II human
+
FY2004 FY2006FY2005
“High Capacity” Top-7 Human Plasma
While Maintaining our Focus:Recovery of sample (fewest number of steps, return of sample)SelectivityReproducibility (run to run, lot to lot of product)Reliability and increased productivity
(April 2006)
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
Selectivity of Plasma-7 ColumnProteins Identified in Bound Fraction by LC/MS/MS
Flow Through Fractions from different amounts of loaded plasma sample
161514
HSA13Alpha1 -Antitrypsin, HSA12
HSA, Transferrin, 11Transferrin, HSA10
IgG, HSA9HSA,8HSA, Haptoglobin, IgG7
IgG, HSA, 6IgG, HSA5
IgG, HSA, IgA4IgG, IgA3
Fibrinogen, , HSA
2Fibrinogen1
Proteins IdentifiedGel Band #
200.0
116.397.4
66.3
55.4
36.5
31.0
21.5
14.4
6.0
kDa
161514
HSA13Alpha1 -Antitrypsin, HSA12
HSA, Transferrin, 11Transferrin, HSA10
IgG, HSA98
HSA, Haptoglobin, IgG7IgG, HSA, 6
IgG, HSA5IgG, HSA, IgA4
IgG, IgA,3
Fibrinogen, Haptoglobin , HSA
2Fibrinogen1
Proteins IdentifiedGel Band #
200.0
116.397.4
66.3
55.4
36.5
31.0
21.5
14.4
6.0
kDa
STD 60 uL 70 uL 80 uL
Human PlasmaBound Fraction
Hemoglobin alpha chainApolipoprotein
Apolipoprotein H
Ceruloplasmin
Complement C3
Complement B pre
Alpha-1-antichymotrypsin
Untargeted Proteins
ELISA analysis indicate 99.4% depletion of Fibrinogen from 60, 70 and 80 μl of a plasma load on a 4.6x100mm column4-4-20%20% SDS SDS -PAGE-PAGE
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
Reproducibility from Run to Run
SDS-PAGE analysis of the flow-through fractions from multiple runs
on a Human Plasma 7 column
Comparison of runs 40, 80, 120, 160, & 200
200.0
116.397.4
66.355.4
36.531.0
21.5
14.46.0
kDa
4-20% SDS-PAGE
1 2 3 4 5 6 7 8 1- Human Plasma2- Mark12 Standards3- Flow-through Fraction, Run 14- Flow-through Fraction, Run 405- Flow-through Fraction, Run 806- Flow-through Fraction, Run 1207- Flow-through Fraction, Run 1608- Flow-through Fraction, Run 200
10 μg of protein/well
200.0
116.397.4
66.355.4
36.531.0
21.5
14.46.0
kDa
4-20% SDS-PAGE
1 40 80 120 160 200 1 2 3 4 5 6 7 8 1- Human Plasma
2- Mark12 Standards3- Flow-through Fraction, Run 14- Flow-through Fraction, Run 405- Flow-through Fraction, Run 806- Flow-through Fraction, Run 1207- Flow-through Fraction, Run 1608- Flow-through Fraction, Run 200
10 μg of protein/well
Column performs reproducibly for 200 runs!
Bound Fraction(Bands excised for confirmation of ID
by MS/MS)
Std
BoundFractionFlow-through
Fraction
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Pub 5989-5136EN
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 separationsReproducibilityHigh sample loading capacity (3X higher than most standard RP columns)Lifetime
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Pub 5989-5136EN
Comparison of mRP with Zorbax SB300-C8
mRP-C18
min0 10 20 30 40 50
mAU
0
5
10
15
20
25
30
Acid-glycoprotein ApolipoproteinA1
complement component C4
hemopexin
SB300-C8
min0 10 20 30 40 50
Norm.
0
10
20
30
40
Abs
orba
nce(
280
nm)
Abs
orba
nce
(280
nm
)
Retention TimeRetention Time
Sample: 270ug flow-through (6M urea/5.0% AcOH) of immunodepleted human serum from Multiple Affinity Removal System columnColumns: 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 280nmMobile 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
Sample: 270ug flow-through (6M urea/5.0% AcOH) of immunodepleted human serum from Multiple Affinity Removal System columnColumns: 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 280nmMobile 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
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
Protein Fractionation on mRP(4.6 x 50mm mRP-C18)
min0 10 20 30 40 50 60 70 80
mAU
0
1
2
3
4
5
6
7mAU
00 10 20 30 40 50 60
1
2
3
4
5
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7
8
HeLa cell lysate (352ug)Hela Membrane Prep
min0 10 20 30 40 50 60
mAU
0
5
10
15
20
25
min0 10 20 30 40 50
mAU
0
5
10
15
20
25
Highest Recovery Lipids
Human Brain membrane lipid Raft prep (500ug)“Top-6” depleted human serum
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Pub 5989-5136EN
min0 10 20 30 40 50 60
LIPID ELUTION
Preliminary LC-MS data of the lipid fraction isolated from the human brain membrane rafts sample
min5 10 15 20 25
0
100000
200000
300000
400000
500000
600000760.5 m/z C34:1 PC
732.5 m/z C32:1 PC
731.5 m/z C18:0 SM
734.5 m/z C32:0 PC
788.5 m/z C36:1 PC
813.6 m/z C24:1 SM
703.5 m/z C16:0 SM
PC = phosphatidylcholineSM = sphingomyelin
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
3
4
5
6
7 128
2
1
11
10
913
Lipid Raft Sample: mRP Fractionation followed by 1D-SDS PAGE Fractionation
Selected Excised Bands Which are Intregal Membrane Proteins1. Voltage-Dependent Anion Selective Channel Protein 12. Cytochrome C Oxidase subunit IV (COX IV)3. Cytochrome C Oxidase subunit IV (COX IV)4. 2’,3’-Cyclic-Nucleotide 3”-Phosphodiesterase (CNP)5. Spectrin Alpha Chain, Brain (Alpha-II Spectrin)6. Vacuolar ATP Synthase Subunit E7. Creatine Kinase, B Chain
8. ATP Synthase alpha chain9. Vacuolar ATP Synthase Subunit D10. Vacuolar ATP Synthase Subunit B11. Contactin Associated Protein12. Vacuolar ATP Synthase Subunit C13. ATP Synthase Chain B14. Thy-1 Membrane Glycoprotein Precursor
(Thy1)
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
Lipid Raft Sample: Reproducibility and Baseline Stability
Overlay of 5 Chromatograms (Lipid Raft Sample)A
bsor
banc
e(28
0 nm
)
Time (min.)
Red – prerun column injection
Blue – postrun column injection after 5 separations
Red – prerun column injection
Blue – postrun column injection after 5 separations
Abs
orba
nce(
280
nm)
Baseline Before and After Sample Injection
Time (min.)
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
4.6 x 50mm mRP
Hela Cell Membrane mRP Fractions followed by 1D-SDS PAGE FractionationHela membranes, starting material, 22 μg
mRP FractionsmRP Fractions
200.0116.3
97.466.355.4
36.531.0
21.5
14.46.0
kDa1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10Gel 1 Gel 2
StdsStds
Lipids
688 364 2863841486,700108HeLaMembranes
PeptidesMatched
688 364 2863841486,700108HeLaMembranes
Sample (216gel bands from mRP)
TotalAcquisitionTime (hrs)
# MS/MS Spectra
Collected
# DistinctPeptidesMatched
# TotalProteinsIdentified
# Membrane ProteinsIdentified
# IntegralMembraneProteinsIdentified
Identification performed by chip-based nano LC/MS/MS from excised gel bands
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
4.6 x 50mm mRP C18 column
M
200.0116.3
97.466.355.4
36.531.0
21.514.4
6.0
kDa 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
200.0116.3
97.466.355.4
36.531.0
21.514.4
6.0
kDa 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 26M
Hela Cell Lysate mRP Fractionation followed by 1D-SDS PAGE Fractionation
mRP ColumnmAU
00 10 20 30 40 50 60
12345678
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Pub 5989-5136EN
For Plasma/Serum Biomarker Discovery: Combine “Top-6”and mRP Fractionation
Removal of 6 most abundant proteins
MARS Immunodepletion
Low abundance proteins
Fractionation of lower abundance proteins
mRP Column Fractionation
Blue Control Serum
Red Cortisol-deficient Serum
Green Rheumatoid Serum
Spectrum MillTwo fractions analyzed by MS tryptic digestion
LC/MSD Trap XCT
Simultaneously:ConcentrateDesaltFractionate
serum # spectra
totalintensity
def. Cort.# spectra
totalintensity
HighRheumatoid
# spectratotal
intensity# UniquePeptides Score Protein
14 0 43.09E+070.00E+00 8.15E+06 12 178.37 H factor 1 (complement)
8 0 91.94E+070.00E+00 2.26E+07 8 115 apolipoprotein H (beta-2-glycoprotein I)
0 3 10.00E+004.65E+06 1.60E+06 3 39.47 ceruloplasmin
0 0 20.00E+000.00E+00 3.54E+06 2 30.34 complement component 1 inhibitor precursor
2 0 08.65E+060.00E+00 0.00E+00 2 28.61 apolipoprotein C-III precursor
1 0 21.84E+060.00E+00 3.13E+06 2 27.34 complement factor B preproprotein
0 0 20.00E+000.00E+00 3.40E+06 2 24.99 hemopexin
0 0 20.00E+000.00E+00 4.13E+06 2 24.77 alpha-1-acid glycoprotein 2 precursor
For Research Use Only. Not for use in diagnostic procedures.
Off-Gel ElectrophoresisTechnology and Applications
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
StrategyTowards Low Abundance Proteins with Immunodepletion & OGE
High-abundanceprotein-free fraction
Enriched, low-abundanceprotein fractions
Crude sample
Fraction I
Fraction II
MARS Separation
Removal
OGE Fractionation
of knownproteins or protein classes (specific)
reducing dynamic range
Fractionation and enrichment of
unknown proteins and peptides (generic)
reducing sample complexity
MARS: Multiple Affinity Removal SystemOGE: Off-Gel Electrophoresis
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
pI-based Fractionation: Off-Gel-Electrophoresis
pH gradient immobilized (IPG gels)
Number of fractions 30
Fraction volume 0.05 - 0.1 ml
Resolution 0.05-0.5 pH
Separation time 5 - 20 h
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
Plasma Proteome WorkflowImmunodepletion, OGE, LC/MS
Workflow and instrumental setup of the immunoaffinity column, OGE and LC/MS system
LC/MS system
LC/MSD Trap XCT2nd pump
Solvent tray
Solvent tray
Degasser
nanoflow pump
6-port micro switchingValve and holder
Cooler
Orthogonal nanospray sourceand nanocolumn
-WPS
Multiple Affinity Removal Column
Off-Gel Electrophoresis
LLLLLLL
HH
LLLHHHHHHH LLLL
HHHHHHHH
LLLLLLLLLLLLLL
HH
LLLHHHHHHH LLLL
HHHHHHHHHHHHHHHH
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
Analysis of OGE Fractions by 1D Electrophoresisfraction # * 1/2 3 4 5 6 7 8 9 10 11 12 13 14 15
E. coli cell extractCoomassie Brillant Blue stain
* unfractionated sample
4
5
pH
6
7
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
Analysis of OGE Fractions by High Resolution 2DE Albumin-Depleted Human Plasma, Silver Stain; Experimentdone 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 #4pH 4.77-4.93
fraction #5pH 4.93-5.10
fraction #6pH 5.10-5.26
fraction #7pH 5.26-5.42
almost no overlap between fractions
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 HeLaS3 Cell Extract* analyzed by Chip-LC/MS
Fraction 1-15, pH 3-10Pr
otein
s sor
ted
by in
crea
sing
calcu
lated
pI
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.
Pub 5989-5136EN
OGE Fractionation of Peptides - E.Coli TrypticDigestAverage Peptide pI with Standard Deviation for Autovalidated Peptides
Auto validated peptides
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
0 5 10 15 20 25
Fraction
calc
pI
pH in centerAverage peptide_pI
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
Bar Chart
F25
2265
674
251
102 43 20 17 6 4 3 1 1 1
1 2 3 4 5 6 7 8 9 10 11 13 160
250
500
750
1000
1250
1500
1750
2000
2250
Fractions
64,6%
19.2%
7.1%2.9%
670 proteins identified with Autovalidation criteria
3454 distinct peptides identified
5840 spectra
=> The majority of peptides are found in only 1 or 2 fractions!
OGE Fractionation of Peptides - E.Coli Tryptic Digest Number of Peptide Identifications in Number of Fractions
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
OGE Fractionation of PeptidesTrypsin Digested E-Coli Lysate
Scatter Plot (2)
delta pI-8 -6 -4 -2 0 2 4 6
5
10
15
20
25
Scatter Plot (2)
delta pI-8 -6 -4 -2 0 2 4 6
5
10
15
20
25
Peptide scores plotted against delta pI (3454 peptides, 5840 spectra, valid after autovalidation)
Peptide scores plotted against delta pI(5251 spectra, not valid after autovalidation)
Can be used to search for charged PTM’s, peptides with high delta pI and high score are possible candidates.
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
OGE Fractionation of PeptidesTrypsin Digested E-Coli Lysate
Scatter Plot (2)
delta pI-8 -6 -4 -2 0 2 4 6
5
10
15
20
25
Peptide scores plotted against delta pI (11091 spectra, valid after autovalidation = green, not validated = red) Scatter Plot (2)
delta pI-8 -6 -4 -2 0 2 4 6
5
10
15
20
25
Peptide scores plotted against delta pI (1964 not validated spectra within ± 0.63 pH of center pH of well)
~35% of spectra not autovalidated are within ± 0.5pH. Using this information, up to 20% more peptides can be identified!
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
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 6.79fraction pH:
Pub 5989-5136EN
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
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.
For Research Use Only. Not for use in diagnostic procedures.
Pub 5989-5136EN
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
For Research Use Only. Not for use in diagnostic procedures.