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Pub 5989-5136EN New Sample Preparation and Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures.

New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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Page 1: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

Pub 5989-5136EN

New Sample Preparation and Protein Fractionation

Techniques

For Research Use Only. Not for use in diagnostic procedures.

Page 2: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 3: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 4: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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

For Research Use Only. Not for use in diagnostic procedures.

Page 5: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

Pub 5989-5136EN

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.

Page 6: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

Why Multiple Affinity Removal System?

Plasma Plasma after Top-6 Depletion

Pub 5989-5136EN

Data: Dr. Y.K. Paik

For Research Use Only. Not for use in diagnostic procedures.

Page 7: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 8: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 9: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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

For Research Use Only. Not for use in diagnostic procedures.

Page 10: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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

For Research Use Only. Not for use in diagnostic procedures.

Page 11: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 12: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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

6

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

For Research Use Only. Not for use in diagnostic procedures.

Page 13: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 14: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 15: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 16: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 17: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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

For Research Use Only. Not for use in diagnostic procedures.

Page 18: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 19: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

Off-Gel ElectrophoresisTechnology and Applications

For Research Use Only. Not for use in diagnostic procedures.

Page 20: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 21: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 22: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 23: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 24: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 25: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 26: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 27: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 28: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 29: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 30: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 31: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 32: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.

Page 33: New Sample Preparation and Protein Fractionation …...Protein Fractionation Techniques For Research Use Only. Not for use in diagnostic procedures. Pub 5989-5136EN What is New in

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.