proteinchip™ protocol: serum protein profiling

ProteinChip™ Protocol:Serum Protein Profiling

Tai-Tung Yip

Christine Yip

Ciphergen Biosystems, Inc.

Palo Alto, CA

Introduction:Serum Protein Profiling Standard Protocol

Objectives Develop a simple protocol for serum protein profiling Achieve optimal resolution of serum proteins using

standard ProteinChipTM arrays Finish whole process within one day Test for robustness and reproducibility

-1 antitrypsin 44324 (60000) pI 5.37 1.8 mg/ml-1 antichymotrypsin 45265.8 (62000) pI 5.32-2 antiplasmin 50450.9 pI 5.87Antithrombin III 49039 (64000) pI 5.95Ceruloplasmin 120085 pI 5.4Clusterin (apo-J) 50062.6 (40000) pI 5.89Clusterin A chain 25883 pI 5.66Complement C3 chain 113028 pI 5.55Complement C3 chain 71332.7 pI 6.82Complement C3 184967.4 pI 6.0Complement C4 chain 33073.6 pI 6.37Complement factor D 23800Connective tissue activating peptide III (CTAP) 9291.7 pI 7.84C-reactive protein 23047 pI 5.28 2-100 ng/mlFerritin 70 ng/ml-1B glycoprotein 51940.7 (72000) pI 5.65Haptoglobulin -1 chain 9192.2 pI 5.23Haptoglobulin -2 chain 15945.8 pI 5.57Haptoglobulin chain 27265 (42000) pI 6.32Haptoglobulin 1 36430 2 mg/mlHemoglobin chain 15126.4 pI 8.73Hemoglobin chain 15867.2 pI 6.81Hemopexin 49295.4 (73000) pI 6.43Histidine-rich glycoprotein 57660 (65000) pI 7.03-2-HS glycoprotein chain A 30221.9 (55000) pI 4.53IgA 3 mg/mlIgG 12 mg/mlIgM 1.5 mg/mlKininogen light chain 28235.7 pI 6.36Leucine-rich -2 glycoprotein (LRG) 34346.4 pI 5.66-2 macroglobulin 160797 pI 5.95

Serum Protein ProfilingMajor identified serum proteins

2.2 mg/ml

-1 microglobulin 20846.7 pI 6.13-2 microglobulin 20 ng/mlMyoglobin 25-45 ng/mlOrosomucoid acid (-1 acid glycoprotein) 21560 (45000) pI 5.0Paraoxonase 39618 (45000) pI 5.08Plasminogen 88300 pI 7.08Serum amyloid P component 23258.5 pI 6.12Serum albumin 66472 pI 5.67 27-39 mg/ml plasmaTransferrin 75181.4 (78000) pI 6.7 3.5 mg/mlTransferrin receptor 2 g/mlTransthyretin 13761.4 (15000, 30000) pI 5.35Troponin I (cardiac) 0.5 ng/mlVitronectin V10 subunit 9266.4 pI 5.08Zinc -2 glycoprotein 32145 (40000) pI 5.58Lipoproteins:HDL 1.7 mole/ml plasmaLDL 4.3mole/ml plasmaVLDL1 1100 g/ml plasmaVLDL2 1030g/ml plasmaIDL 1256g/ml plasmaApolipoprotein A-I 28078.6 pI 5.27 1-1.5 mg/ml plasmaApolipoprotein A-II 8707.9 pI 5.05Apolipoprotein A-IV 43374.5 pI 5.2Apolipoprotein B100 70 g/ml plasmaApolipoprotein C-III 8764.7 pI 4.72 17 g/ml plasmaApolipoprotein C-II 8914.9 pI 4.66Apolipoprotein D 19303, 26-30000 pI 5.2Apolipoprotein E 34236.7 pI 5.52 45 g/ml

Serum Protein ProfilingMajor identified serum proteins

Serum Protein ProfilingSchematic

Pool

Recover sampleWash

Cibacron blue

Human serum

Mixed (MM1)Cationic (SAX2)

Wash 1 Wash 2

Ni(II) (IMAC3)

Wash 1 Wash 2 Wash 1 Wash 2

Hydrophobic (H4)

Wash 1 Wash 2

Serum Protein ProfilingI. Dilution and serum albumin removal

Spin column700 xg 30 sec

Save filtrate in the eppendorf tube

Put column into a fresh eppendorf tube.Vortex in the cold

15 min

Add 50 ul diluted serum sample to column

Prepare Cibacron blue spin column

(100 ul 50% (v/v) bead suspensionequilibrated with 3x300 ul of 1M urea, 0.125% CHAPS, PBS.

Spin column dry at 700 xg 30 sec each time.)

Vortex in the cold10 min

Human serum20 ul

+30 ul 8M urea (Sigma electrophoresis grade)1% CHAPS (Sigma), PBS

Final volume~150 ul

Spin column700 xg 30 sec

Collect filtrate in the same eppendorf tube

Vortex in the cold15 min

Add 100 ul1M urea 0.125% CHAPS, PBS

to the column

Serum Protein ProfilingI. Dilution and serum albumin removal

50000 100000 150000

0

5

10

6739

9.4

0

0.5

1

1.5

6743

3.1

0

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4

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5

10

15

Serum Protein ProfilingI. Serum albumin removal

Sample: 1:1.5 8M urea 1% CHAPS

15 mM HEPES

Cibacron Blue column:1M urea, 0.12% CHAPS

PBS

HSAFraction

1

2

3

Bead

1

Next page



15 mM HEPES


PBS

Fraction1

2

3

Bead

10000 20000 30000 40000

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6738

7.9

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2

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20

30


15 mM HEPES


PBS

HSA Fraction1

2

3

Bead

2

Repeat


15 mM HEPES


PBS

50000 100000 150000

0

5

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15

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5

10

15

0

5

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15

0

5

10

15

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5

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15

Fraction1

2

Bead

High Salt Wash

50% Ethylene Glycol Wash

HSA


To check for recovery of

proteinstightly bound

to bead


50000 100000 150000

0

10

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30

0

2.5

5

7.5

10

0

1

2

3

0

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40


15 mM HEPES


50 mM HEPES

HSAFraction

1

2

3

Bead

No salt

Poor recovery of some proteins


50000 100000 150000

0

2

4

6

6752

2

0

2.5

5

7.5

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7.5

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4

Sample: 1:1.5 1% Triton X100PBS + 0.85M NaCl

Cibacron Blue column:0.1% Triton X100

PBS + 0.15 M NaCl

HSA Fraction1

2

3

Bead

Too much salt

Poor capture of serum albumin

50000 100000 150000

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1

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4

50000 100000 150000

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4

50000 100000 150000

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7.5

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7.5

Sample: 1:1.5 1% Triton X1001% dodecyl maltoside

1%CHAPSPBS

Cibacron Blue column:0.1% Triton X100

0.1% dodecyl maltoside0.1%CHAPS

PBS

Fraction1

2

3

Bead

HSA


No urea

Poor recovery of some proteins

Add 5 ul of 50 mM HEPES to each spot

Add 5 ul of diluted and extracted serum sample to spots 1, 2, 3, 4

Repeat with fresh buffer

Mix at room temperature for 5 min

Draw hydrophobic ring around each spot

Remove the samples from SAX2 chip

Add 5 ul of 50 mM HEPES to spots 1, 2Add 5 ul of 1 M urea 0.125% CHAPS, 0.25 M NaCl, 50 mM HEPES to spots 3, 4

Serum Protein ProfilingII. ProteinChipTM array Cationic site (SAX2)

Mix at room temperature for 15 min in humid chamber (OR at least 30 min without mixing)


Repeat process

Wash chip with large volume of water


Air dry chip

Add 0.4 ul of SPA to spots 1, 3 and 0.4 ul of CHCA to spots 2, 4

Remove the washes from the spotsAdd fresh 5 ul of 50 mM HEPES to spots 1, 2.Add fresh 5 ul of 1 M urea 0.125% CHAPS, 0.25 M NaCl, 50 mM HEPES to spots 3, 4.




1 2 3 4 6 7 85

ProteinChipTM array1 2 3 4 6 7 85

Draw hydrophobic rings

1 2 3 4 6 7 85

Add 5 ul equilibration buffer to each spot

5 min mixing

Remove bufferRepeat


1 2 3 4 6 7 85

Add 5 ul sample to each spot.

Incubate in humid chamber with mixing 15 min (OR at least 30 min without mixing)

Remove samples.

1 2 3 4 6 7 85

Add 5 ul wash buffer 1 or 2.


Incubate in humid chamber with mixing 5 min (OR at least 15 min without mixing)1 2 3 4 6 7 85

Add 5 ul fresh wash buffer 1 or 2.Discard washes.

Incubate in humid chamber with mixing 5 min (OR at least 15 min without mixing)


Bulk water wash of each spot

Air dry

Add 0.4 ul of SPA to spots 1, 3, 5, 7 and0.4 ul of CHCA to spots 2, 4, 6, 8

Repeat process

Repeat with fresh Ni(II)


Add 5 ul of 50 mM Ni(II) to each spot

Draw hydrophobic ring around each spot

Repeat with fresh buffer


Add 5 ul of PBS to each spot

Serum Protein ProfilingIII. ProteinChipTM array Ni(II) site (IMAC3)

Add 5 ul of diluted and extracted serum sample to spots 1, 2, 3, 4


Bulk wash with water

Repeat process


Remove the samples from the spots of Ni(II) chip

Add 5 ul of PBS to spots 1, 2.Add 5 ul of 1 M urea 0.125% CHAPS, 100 mM acetate, 0.5 M

NaCl, pH 4.5 to spots 3, 4.

Remove the washes from the spots of Ni(II) chip. Add fresh 5 ul of PBS to spots 1, 2.

Add fresh 5 ul of 1 M urea 0.125% CHAPS, 100 mM acetate, 0.5 M NaCl, pH 4.5 to spots 3, 4.




Wash chip with large volume of water

Air dry chip



Repeat with spots 2, 3, 4

Add 1 ul of diluted and extracted serum sample to spot 1 before acetonitrile is completely dry

Add 0.5 ul of acetonitrile to spot 1

Air dry sample

Add 5 ul of water to spots 1, 2Add 5 ul of 30% isopropanol/acetonitrile (2:1) to spots 3, 4

Repeat with fresh washes

Remove wash solutions

Serum Protein ProfilingIV. ProteinChipTM array Hydrophobic site (H4)


Air dry chip

Serum Protein ProfilingIV. ProteinChipTM array Hydrophobic site (H4)



Serum Protein ProfilingV. ProteinChipTM array Mixed Mode site (MM1)

Repeat with spots 2-4

Add 5 ul of diluted and extracted serum sample to spots 1 before acetonitrile is completely dry

Add 0.5 ul of acetonitrile to spot 1

Remove samples from spots of MM1 chip

Add 5 ul of 50 mM HEPES to spots 1, 2Add 5 ul of 100 mM sodium acetate pH 4 to spots 3, 4



Serum Protein ProfilingV. ProteinChipTM array Mixed Mode site (MM1)

Repeat process

Wash chips with large volume of water

Air dry chip

Remove the wash solutions from the spots of MM1. Add fresh 5 ul of 50 mM HEPES to spots 1, 2

Add fresh 5 ul of 100 mM sodium acetate pH 4 to spots 3, 4




50000 100000 150000

Sample: Cibacron blue spin column 1

Pool

K70 spin column 1M urea, 0.125% CHAPS

PBS(Drain column dry,

add 30 ul sample carefullyto top of gel,

spin 700 xg 4sec,collect filtrate,

add 30 ul same buffer,spin,

collect filtrate,repeat elution if necessary.)

Fraction1

2

3

Serum Protein ProfilingVI. Size separation


Pool

SuperDex75 spin column1M urea, 0.125% CHAPS

PBS


50000 100000 150000

Fraction1

2

3

4

5

6



Pool

SuperDex75 spin column1M urea, 0.125% CHAPS

PBS

5000 10000 15000 20000

Fraction1

2

3

4

5

6

2000 4000 6000 8000

0

25

50

75

1006.1+H

1084.3+H

1282.8+H

1638.2+H

3163.7+H

5814.8+H

6962.1+H

Calibrated Externally on 11/09/98 02:20 from Data Average w hich is Calibrated Internally on 11/09/98 02:20 using 6963.5+1H, 5807.7+1H, 3160.7+1H, 1637.9+1H, 1281.4+1H, 1084.25+1H

Peptide standards(CHCA)

20000 40000 60000 80000

0

10

2012360.9+H

16961.7+H

66458.4+H


Protein standards(SPA)

2000 4000 6000 8000

0

20

40

1084.0+H

1281.5+H

1637.4+H

3162.2+H

5808.4+H

6962.0+H

Calibrated Internally on 11/09/98 02:20 using 6963.5+1H, 5807.7+1H, 3160.7+1H, 1637.9+1H, 1281.4+1H, 1084.25+1H

20000 40000 60000 80000

0

10

20

30

40

12384.9+H

16980.2+H

66545.2+H


At least duplicate to establish reproducibility. Recalibrate if necessary.

Serum Protein ProfilingVII. Reading ProteinChipTM array 1. Check instrument performance

Serum Protein ProfilingVII. Reading ProteinChipTM array 2. Read samples manually

5000 10000 15000

0

2.5

5

7.5

10

Calibrated Externally on 11/09/98 07:46 from pepstd 1 w hich is Calibrated Internally on 11/09/98 07:46 using 6963.5+1H, 5807.7+1H, 3160.7+1H, 1637.9+1H, 1281.4+1H, 1084.25+1H

Low laser energy(Sensitivity 6, Intensity 10-25)

Average of 50 laser shots

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7.5

10


50000 100000 150000

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2.5

5

7.5


High laser energy(Neutral density filter out, Sensitivity 9, Intensity 45)

Average of at least 80 laser shots

5000 10000 15000

0

2.5

5

7.5


At least duplicate to establish reproducibility

0 50000 100000 150000

0

25

50

75

100

Calibrated Externally on 12/11/98 11:30 from pepstd.spe w hich is Calibrated Internally on 11/30/98 10:17 using 6963.5+1H, 3160.7+1H, 1637.9+1H, 1084.25+1H, 1007.2+1H

Spot Protocol:1: Set high mass to 200000 Daltons, optimized from 40000 Daltons to 100000 Daltons.2: Set starting laser intensity to 35.3: Set starting detector sensitivity to 9.4: Set laser NDF Out (more light).5: Set data acquistion method to Automatic Laser Adjustment6: Set shots to collect to 75 shots.7: Set points on scale to accept to 3 points and On-scale intensity to 26 (10%).8: Set points off scale to reject to 2 points and Off-scale intensity to 179 (70%).9: Increase laser intensity by 4% after 3 consecutive low shots.10: Decrease laser intensity by 4% after 1 consecutive high shots.11: Revive signal with increased laser after 8 consecutive shots with out signal, boost intensity 15%.12: Set minimum number of shots per fresh spot to 8 shots.13: Process sample.

Serum Protein ProfilingVII. Reading ProteinChipTM array 3. Read samples by Automode

5000 10000 15000

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Wash 1

Wash 2

SPA

SPA

CHCA

CHCA

Low laser energy

Serum Protein ProfilingVIII. Representative profiles - SAX2

50000 100000 150000

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2.5

5

7.5

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1

2

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0.75

Wash 1

Wash 2

SPA

SPA

CHCA

CHCA

High laser energy

Serum Protein ProfilingVIII. Representative profiles - SAX2

Wash 1

Wash 2

Low laser energy

Serum Protein ProfilingVIII. Representative profiles - Ni(II)

CHCA

CHCA

5000 10000 15000

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0.5

1

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1

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2

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5000 10000 15000

SPA

SPA

50000 100000 150000

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12.5

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3

50000 100000 150000

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Wash 1

Wash 2

SPA

SPA

CHCA

CHCA

High laser energy

Serum Protein ProfilingVIII. Representative profiles - Ni(II)

Serum Protein ProfilingVIII. Representative profiles - H4

Wash 1

Wash 2

SPA

CHCA

CHCA

SPALow laser energy

5000 10000 15000 20000

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2.5

5

7.5

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6

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30

40

0

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20

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Serum Protein ProfilingVIII. Representative profiles - H4

50000 100000 150000

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0.1

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0.1

0.15

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50000 100000 150000

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Wash 1

Wash 2

SPA

CHCA

SPA

CHCA

High laser energy

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2

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Wash 1

Wash 2

SPA

SPA

CHCA

CHCA

Low laser energy

Serum Protein ProfilingVIII. Representative profiles - MM1 (stainless steel)

Serum Protein ProfilingVIII. Representative profiles - MM1 (aluminum)

Low laser energy

5000 10000 150005000 10000 15000

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50 mM HEPES

100 mM acetate pH 4

30% ethylene glycol

10% acetonitrile 0.1% triethylamine

50000 100000 150000

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50000 100000 150000

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High laser energy

Wash 1

Wash 2

SPA

SPA

CHCA

CHCA

Serum Protein ProfilingVIII. Representative profiles - MM1 (stainless steel)

Serum Protein ProfilingVIII. Representative profiles - MM1

50000 100000 150000

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HEPES

1M urea 0.1%CHAPSHEPES/NaCl

5% acetonitrile0.1% TFA

5% acetonitrile0.1% TEA

High laser energy

50000 100000 150000

50000 100000 150000

910(1-12.5u1pbs) cblue nc

910(1-12.5u1pbs) cblue nc 1



1. Arrange data of one spot into Experiment

0 50000 100000 150000

0 50000 100000 150000



2. Detect peaks (combine useful data from all spectra)

Low mass

High mass

Serum Protein ProfilingIX. Data Processing

3. Arrange peak maps of 4 spots (4 ProteinChipTM sites) into Experiment

0 5000 10000 15000

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75

50000 100000 150000

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75

4. Combine Peak maps

5000 10000 15000

5000 10000 15000

910(1-12.5u1pbs) cblue 3

910(1-12.5u1pbs) cblue cs 6


910(u1pbs) t tc low

Low mass

50000 100000 150000

50000 100000 150000

910(1-12.5u1pbs) cblue 1

910(1-12.5u1pbs) cblue cs 3


910(u1pbs) t tc high

High mass


Disease combine

Normal combine

Disease Normalcompare

Disease spot 1Disease spot 2Normal spot 1Normal spot 2

0

2

4

0

2.5

5

7.5

10

4000 6000 8000 10000-7.5

-5

-2.5

0

2.5

Peak count:169

164

0.05% mass error

Almost no common peaks (no red)!


4000 6000 8000 10000

4000 6000 8000 10000

7 cblue wg cs 2

7 cblue wg cs(2) 3

10 cblue wg cs 3

10 cblue wg cs(2) 2

7 cs combine

10 cs combine

7 10 cs compare

0.15% mass error

148

135

Still too many unique peaks

CONFIDENTIAL


4000 6000 8000 10000

4000 6000 8000 10000

7 cblue wg cs 2

7 cblue wg cs(2) 3

10 cblue wg cs 3

10 cblue wg cs(2) 2

7 cs combine

10 cs combine

7 10 cs compare

0.3% mass error

125

110

Reasonable

Too high mass errorleading to counting 2 or more peaks as one


Serum Protein ProfilingX. PeakMaps

5000 10000 15000

SAX2

Ni(II)

H4

SCX1

Combine

S/N 100.5% mass error

Serum Protein ProfilingX. PeakMaps

50000 100000 150000

SAX2

Ni(II)

H4

AS1

Combine

S/N 100.5% mass error

Serum Protein ProfilingXI. Reproducibility

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SAX2SPA

Low laser energy4 experiments

Cibacron blue spin column 1



50000 100000 150000

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SAX2SPA

High laser energy4 experiments




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Ni(II)CHCA

Low laser energy4 experiments

Conclusions:Serum Protein Profiling Protocol

Sample treatment Recommendation - use urea and detergent to disrupt protein-

protein interactions Recommendation - use Cibacron blue spin column to remove

most of serum albumin Size spin column optional

Profiling protocol 4 standard ProteinChipTM arrays with 2 standard washes for each

array / two EAMS / automode data collection with two laser energy levels / auto peak picking

Performance Adequate resolution - approaching 300 ProteinPeak at s/n of 10

and 0.5% mass error Need to prove robustness and reproducibility

proteinchip™ protocol: serum protein profiling

Documents

serum proteinsa

serum albumin removalsample

serum albumin removaladd

pbsserum protein profilingi

columnserum protein

ul1m urea

hepescibacron blue column

ul diluted serum sample