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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 - PowerPoint PPT Presentation
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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
2
4
0
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
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
Fraction1
2
3
Bead
10000 20000 30000 40000
0
5
10
15
0
1
2
3
4
0
1
2
0
10
20
30
Serum Protein ProfilingI. Serum albumin removal
50000 100000 150000
0
5
10
15
0
0.25
0.5
0.75
6738
7.9
0
0.5
1
1.5
2
0
10
20
30
Sample: 1:1.5 8M urea 1% CHAPS
15 mM HEPES
Cibacron Blue column:1M urea, 0.12% CHAPS
PBS
HSA Fraction1
2
3
Bead
2
Repeat
Sample: 1:1.5 8M urea 1% CHAPS
15 mM HEPES
Cibacron Blue column:1M urea, 0.12% CHAPS
PBS
50000 100000 150000
0
5
10
15
0
5
10
15
0
5
10
15
0
5
10
15
0
5
10
15
Fraction1
2
Bead
High Salt Wash
50% Ethylene Glycol Wash
HSA
Serum Protein ProfilingI. Serum albumin removal
To check for recovery of
proteinstightly bound
to bead
Serum Protein ProfilingI. Serum albumin removal
50000 100000 150000
0
10
20
30
0
2.5
5
7.5
10
0
1
2
3
0
20
40
Sample: 1:1.5 8M urea 1% CHAPS
15 mM HEPES
Cibacron Blue column:1M urea, 0.12% CHAPS
50 mM HEPES
HSAFraction
1
2
3
Bead
No salt
Poor recovery of some proteins
Serum Protein ProfilingI. Serum albumin removal
50000 100000 150000
0
2
4
6
6752
2
0
2.5
5
7.5
10
0
2.5
5
7.5
0
2
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
0
1
2
3
4
50000 100000 150000
0
1
2
3
4
50000 100000 150000
0
2.5
5
7.5
50000 100000 150000
0
2.5
5
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
Serum Protein ProfilingI. Serum albumin removal
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)
Mix at room temperature for 5 min in humid chamber (OR at least 15 min without mixing)
Repeat process
Wash chip with large volume of water
Serum Protein ProfilingII. ProteinChipTM array Cationic site (SAX2)
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.
Mix at room temperature for 5 min in humid chamber (OR at least 15 min without mixing)
Mix at room temperature for 5 min in humid chamber (OR at least 15 min without mixing)
Serum Protein ProfilingII. ProteinChipTM array Cationic site (SAX2)
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
Serum Protein ProfilingII. ProteinChipTM array Cationic site (SAX2)
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.
Serum Protein ProfilingII. ProteinChipTM array Cationic site (SAX2)
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)
Serum Protein ProfilingII. ProteinChipTM array Cationic site (SAX2)
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)
Mix at room temperature for 10 min
Add 5 ul of 50 mM Ni(II) to each spot
Draw hydrophobic ring around each spot
Repeat with fresh buffer
Mix at room temperature for 5 min
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
Mix at room temperature for 15 min in humid chamber (OR at least 30 min without mixing)
Bulk wash with water
Repeat process
Serum Protein ProfilingIII. ProteinChipTM array Ni(II) site (IMAC3)
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.
Mix at room temperature for 5 min in humid chamber (OR at least 15 min without mixing)
Mix at room temperature for 5 min in humid chamber (OR at least 15 min without mixing)
Mix at room temperature for 5 min in humid chamber (OR at least 15 min without mixing)
Wash chip with large volume of water
Air dry chip
Serum Protein ProfilingIII. ProteinChipTM array Ni(II) site (IMAC3)
Add 0.4 ul of SPA to spots 1, 3 and 0.4 ul of CHCA to spots 2, 4
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)
Mix at room temperature for 5 min in humid chamber (OR at least 10 min without mixing)
Air dry chip
Serum Protein ProfilingIV. ProteinChipTM array Hydrophobic site (H4)
Add 0.4 ul of SPA to spots 1, 3 and 0.4 ul of CHCA to spots 2, 4
Mix at room temperature for 5 min in humid chamber (OR at least 10 min without mixing)
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
Mix at room temperature for 15 min in humid chamber (OR at least 30 min without mixing)
Mix at room temperature for 5 min in humid chamber (OR at least 15 min without mixing)
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
Add 0.4 ul of SPA to spots 1, 3 and 0.4 ul of CHCA to spots 2, 4
Mix at room temperature for 5 min in humid chamber (OR at least 15 min without mixing)
Mix at room temperature for 5 min in humid chamber (OR at least 15 min without mixing)
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
Sample: Cibacron blue spin column 2
Pool
SuperDex75 spin column1M urea, 0.125% CHAPS
PBS
Serum Protein ProfilingVI. Size separation
50000 100000 150000
Fraction1
2
3
4
5
6
Serum Protein ProfilingVI. Size separation
Sample: Cibacron blue spin column 2
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
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
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
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
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
50000 100000 150000
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
50000 100000 150000
0
2.5
5
7.5
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
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
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
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
0
2
4
6
0
1
2
3
0
1
2
3
0
0.5
1
1.5
2
Wash 1
Wash 2
SPA
SPA
CHCA
CHCA
Low laser energy
Serum Protein ProfilingVIII. Representative profiles - SAX2
50000 100000 150000
0
2.5
5
7.5
0
5
10
15
0
1
2
0
0.25
0.5
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
0
0.5
1
0
5
10
15
0
0.5
1
1.5
2
0
2.5
5
7.5
10
5000 10000 15000
SPA
SPA
50000 100000 150000
0
2.5
5
7.5
10
12.5
0
2
4
6
0
1
2
3
50000 100000 150000
0
1
2
3
4
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
0
2.5
5
7.5
0
2
4
6
0
10
20
30
40
0
10
20
5000 10000 15000 20000
Serum Protein ProfilingVIII. Representative profiles - H4
50000 100000 150000
0
0.1
0.2
0.3
0.4
0
0.05
0.1
0.15
0
1
2
50000 100000 150000
0
0.1
0.2
0.3
Wash 1
Wash 2
SPA
CHCA
SPA
CHCA
High laser energy
5000 10000 15000
0
1
2
3
4
0
0.5
1
1.5
2
0
2
4
6
0
0.5
1
1.5
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
0
0.2
0.4
0
0.25
0.5
0.75
0
0.5
1
0
0.05
0.1
0.15
0.2
50 mM HEPES
100 mM acetate pH 4
30% ethylene glycol
10% acetonitrile 0.1% triethylamine
50000 100000 150000
0
0.5
1
0
0.2
0.4
0.6
0
1
2
50000 100000 150000
0
0.2
0.4
0.6
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
0
0.5
1
0
0.2
0.4
0
0.5
1
1.5
50000 100000 150000
0
0.02
0.04
0.06
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
910(1-12.5u1pbs) cblue nc 3
910(1-12.5u1pbs) cblue nc 4
1. Arrange data of one spot into Experiment
0 50000 100000 150000
0 50000 100000 150000
910(1-12.5u1pbs) cblue nc 6
910(1-12.5u1pbs) cblue nc 2
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
0
25
50
75
50000 100000 150000
0
25
50
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(1-12.5u1pbs) cblue nc 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(1-12.5u1pbs) cblue nc 2
910(u1pbs) t tc high
High mass
Serum Protein ProfilingIX. Data Processing
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)!
Serum Protein ProfilingIX. Data Processing
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
Serum Protein ProfilingIX. Data Processing
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 ProfilingIX. Data Processing
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
5000 10000 15000 20000
0
1
2
0
5
10
15
0
2
4
6
0
2.5
5
7.5
10
SAX2SPA
Low laser energy4 experiments
Cibacron blue spin column 1
Cibacron blue spin column 2
Serum Protein ProfilingXI. Reproducibility
50000 100000 150000
0
2
4
0
5
10
0
5
10
15
0
2.5
5
7.5
10
SAX2SPA
High laser energy4 experiments
Cibacron blue spin column 1
Cibacron blue spin column 2
Serum Protein ProfilingXI. Reproducibility
5000 10000 15000 20000
0
10
20
0
2.5
5
7.5
0
5
10
15
0
1
2
3
Cibacron blue spin column 1
Cibacron blue spin column 2
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
Recommended