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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

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ProteinChip™ Protocol: Serum Protein Profiling

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  1. ProteinChip™ Protocol:Serum Protein Profiling Tai-Tung Yip Christine Yip Ciphergen Biosystems, Inc. Palo Alto, CA

  2. 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

  3. Serum Protein ProfilingMajor identified serum proteins a -1 antitrypsin 44324 (60000) pI 5.37 1.8 mg/ml 2.2 mg/ml a -1 antichymotrypsin 45265.8 (62000) pI 5.32 a -2 antiplasmin 50450.9 pI 5.87 Antithrombin III 49039 (64000) pI 5.95 Ceruloplasmin 120085 pI 5.4 Clusterin (apo-J) 500 62.6 (40000) pI 5.89 Clusterin A chain 25883 pI 5.66 a Complement C3 chain 113028 pI 5.55 b Complement C3 chain 71332.7 pI 6.82 Complement C3 184967.4 pI 6.0 g Complement C4 chain 33073.6 pI 6.37 Complement factor D 23800 Connective tissue activating peptide III (CTAP) 9291.7 pI 7.84 C-reactive protein 23047 pI 5.28 2-100 ng/ml Ferritin 70 ng/ml a -1B glycoprotein 51940.7 (72000) pI 5.65 a Haptoglobulin -1 chain 9192.2 pI 5.23 a Haptoglobulin -2 chain 15945.8 p I 5.57 b Haptoglobulin chain 27265 (42000) pI 6.32 Haptoglobulin 1 36430 2 mg/ml a Hemoglobin chain 15126.4 pI 8.73 b Hemoglobin chain 15867.2 pI 6.81 Hemopexin 49295.4 (73000) pI 6.43 Histidine-rich glycoprotein 57660 (65000) pI 7. 03 a -2-HS glycoprotein chain A 30221.9 (55000) pI 4.53 IgA 3 mg/ml IgG 12 mg/ml IgM 1.5 mg/ml Kininogen light chain 28235.7 pI 6.36 a Leucine-rich -2 glycoprotein (LRG) 34346.4 pI 5.66 a -2 macroglobulin 160797 pI 5.95

  4. a 20846.7 pI 6.13 -1 microglobulin b -2 microglobulin 20 ng/ml Myoglobin 25-45 ng/ml a Orosomucoid acid ( -1 acid glycoprotein) 21560 (45000) pI 5.0 Paraoxonase 39618 (45000) pI 5.08 Plasminogen 88300 pI 7.08 Serum amyloid P component 23258.5 pI 6.12 Serum albumin 66472 pI 5.67 27-39 mg/ml plasma Transferrin 3.5 mg/ml 75181.4 (78000) pI 6.7 m Transferrin receptor 2 g/ml Transthyretin 13761.4 (15000, 30000) pI 5.35 0.5 ng/ml Troponin I (cardiac) Vitronectin V10 subunit 9266.4 pI 5.08 a Zinc -2 glycoprotein 32145 (40000) pI 5.58 Lipoproteins: m plasma VLDL1 1100 g/ml Apolipoprotein A-II 8707.9 pI 5.05 m plasma HDL 1.7 mole/ml Apolipoprotein A-IV 43374.5 pI 5.2 m Apolipoprotein B100 70 g/ml plasma plasma m VLDL2 1030 g/ml m plasma LDL 4.3 mole/ml m Apolipoprotein C-III 8764.7 pI 4.72 17 g/ml plasma Apolipoprotein C-II 8914.9 pI 4.66 plasma m IDL 1256 g/ml Apolipoprotein D 19303, 26-30000 pI 5.2 m Apolipoprotein E 34236.7 pI 5.52 45 g/ml plasma Apolipoprotein A-I 28078.6 pI 5.27 1-1.5 mg/ml Serum Protein ProfilingMajor identified serum proteins

  5. Pool Serum Protein ProfilingSchematic Human serum Cibacron blue Recover sample Wash Hydrophobic (H4) Mixed (MM1) Cationic (SAX2) Ni(II) (IMAC3) Wash 2 Wash 1 Wash 2 Wash 1 Wash 2 Wash 1 Wash 1 Wash 2

  6. (100 ul 50% (v/v) bead suspension equilibrated with 3x300 ul of 1M urea, 0.125% CHAPS, PBS. Spin column dry at 700 xg 30 sec each time.) Serum Protein ProfilingI. Dilution and serum albumin removal Human serum 20 ul +30 ul 8M urea (Sigma electrophoresis grade) 1% CHAPS (Sigma), PBS Vortex in the cold 10 min Prepare Cibacron blue spin column Add 50 ul diluted serum sample to column Put column into a fresh eppendorf tube. Vortex in the cold 15 min Spin column 700 xg 30 sec Save filtrate in the eppendorf tube

  7. Vortex in the cold 15 min Spin column 700 xg 30 sec Collect filtrate in the same eppendorf tube Final volume ~150 ul Serum Protein ProfilingI. Dilution and serum albumin removal Add 100 ul 1M urea 0.125% CHAPS, PBS to the column

  8. Serum Protein ProfilingI. Serum albumin removal HSA Sample: 1:1.5 8M urea 1% CHAPS 15 mM HEPES Cibacron Blue column: 1M urea, 0.12% CHAPS PBS Fraction 1 10 67399.4 5 0 1 1.5 67433.1 2 1 0.5 0 4 3 2 0 15 Bead 10 5 0 50000 100000 150000 Next page

  9. 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 Fraction 1 15 10 5 0 4 3 2 2 1 0 2 3 1 0 30 Bead 20 10 0 10000 20000 30000 40000

  10. Serum Protein ProfilingI. Serum albumin removal Repeat 15 HSA Fraction 1 Sample: 1:1.5 8M urea 1% CHAPS 15 mM HEPES Cibacron Blue column: 1M urea, 0.12% CHAPS PBS 10 5 2 0 67387.9 0.75 2 0.5 0.25 0 2 1.5 3 1 0.5 0 30 Bead 20 10 0 50000 100000 150000

  11. Serum Protein ProfilingI. Serum albumin removal HSA 15 Fraction 1 Sample: 1:1.5 8M urea 1% CHAPS 15 mM HEPES Cibacron Blue column: 1M urea, 0.12% CHAPS PBS 10 5 0 15 10 2 5 0 15 High Salt Wash 10 To check for recovery of proteins tightly bound to bead 5 0 15 50% Ethylene Glycol Wash 10 5 0 15 Bead 10 5 0 50000 100000 150000

  12. Serum Protein ProfilingI. Serum albumin removal 30 Sample: 1:1.5 8M urea 1% CHAPS 15 mM HEPES Cibacron Blue column: 1M urea, 0.12% CHAPS 50 mM HEPES Fraction 1 HSA 20 10 0 10 2 7.5 5 2.5 0 3 No salt 3 2 1 0 Poor recovery of some proteins Bead 40 20 0 50000 100000 150000

  13. Serum Protein ProfilingI. Serum albumin removal 67522 Sample: 1:1.5 1% Triton X100 PBS + 0.85M NaCl Cibacron Blue column: 0.1% Triton X100 PBS + 0.15 M NaCl HSA 6 Fraction 1 4 2 0 10 2 7.5 5 2.5 0 Too much salt 7.5 3 5 2.5 Poor capture of serum albumin 0 4 Bead 2 0 50000 100000 150000

  14. 4 3 2 1 0 50000 100000 150000 4 3 2 1 0 7.5 50000 100000 150000 5 2.5 0 50000 100000 150000 7.5 5 2.5 0 50000 100000 150000 Serum Protein ProfilingI. Serum albumin removal Sample: 1:1.5 1% Triton X100 1% dodecyl maltoside 1%CHAPS PBS Cibacron Blue column: 0.1% Triton X100 0.1% dodecyl maltoside 0.1%CHAPS PBS HSA Fraction 1 2 3 No urea Bead Poor recovery of some proteins

  15. Serum Protein ProfilingII. ProteinChipTM array Cationic site (SAX2) Draw hydrophobic ring around each spot Add 5 ul of 50 mM HEPES to each spot Mix at room temperature for 5 min Repeat with fresh buffer 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) Remove the samples from SAX2 chip Add 5 ul of 50 mM HEPES to spots 1, 2 Add 5 ul of 1 M urea 0.125% CHAPS, 0.25 M NaCl, 50 mM HEPES to spots 3, 4

  16. Serum Protein ProfilingII. ProteinChipTM array Cationic site (SAX2) Mix at room temperature for 5 min in humid chamber (OR at least 15 min without mixing) Remove the washes from the spots Add 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) Repeat process 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 Add 0.4 ul of SPA to spots 1, 3 and 0.4 ul of CHCA to spots 2, 4

  17. 2 4 3 6 8 5 1 7 ProteinChipTM array 2 3 4 6 8 5 1 7 Draw hydrophobic rings 2 3 4 6 8 5 1 7 Add 5 ul equilibration buffer to each spot 5 min mixing Repeat Remove buffer Serum Protein ProfilingII. ProteinChipTM array Cationic site (SAX2)

  18. 2 4 3 6 8 5 1 7 Incubate in humid chamber with mixing 15 min (OR at least 30 min without mixing) 2 4 3 6 8 5 1 7 Serum Protein ProfilingII. ProteinChipTM array Cationic site (SAX2) Add 5 ul sample to each spot. Remove samples. Add 5 ul wash buffer 1 or 2.

  19. Incubate in humid chamber with mixing 5 min (OR at least 15 min without mixing) 2 4 3 6 8 5 1 7 Incubate in humid chamber with mixing 5 min (OR at least 15 min without mixing) Serum Protein ProfilingII. ProteinChipTM array Cationic site (SAX2) Discard washes. Add 5 ul fresh wash buffer 1 or 2.

  20. Repeat process Bulk water wash of each spot Air dry Add 0.4 ul of SPA to spots 1, 3, 5, 7 and 0.4 ul of CHCA to spots 2, 4, 6, 8 Serum Protein ProfilingII. ProteinChipTM array Cationic site (SAX2)

  21. Mix at room temperature for 15 min in humid chamber (OR at least 30 min without mixing) Serum Protein ProfilingIII. ProteinChipTM array Ni(II) site (IMAC3) Draw hydrophobic ring around each spot Add 5 ul of 50 mM Ni(II) to each spot Mix at room temperature for 10 min Repeat with fresh Ni(II) Bulk wash with water Add 5 ul of PBS to each spot Mix at room temperature for 5 min Repeat with fresh buffer Add 5 ul of diluted and extracted serum sample to spots 1, 2, 3, 4

  22. 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) 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. Repeat process

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

  24. Add 5 ul of water to spots 1, 2 Add 5 ul of 30% isopropanol/acetonitrile (2:1) to spots 3, 4 Mix at room temperature for 5 min in humid chamber (OR at least 10 min without mixing) Serum Protein ProfilingIV. ProteinChipTM array Hydrophobic site (H4) Add 0.5 ul of acetonitrile to spot 1 Add 1 ul of diluted and extracted serum sample to spot 1 before acetonitrile is completely dry Repeat with spots 2, 3, 4 Air dry sample Remove wash solutions Repeat with fresh washes

  25. Mix at room temperature for 5 min in humid chamber (OR at least 10 min without mixing) Serum Protein ProfilingIV. ProteinChipTM array Hydrophobic site (H4) Air dry chip Add 0.4 ul of SPA to spots 1, 3 and 0.4 ul of CHCA to spots 2, 4

  26. 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) Add 0.5 ul of acetonitrile to spot 1 Add 5 ul of diluted and extracted serum sample to spots 1 before acetonitrile is completely dry Repeat with spots 2-4 Remove samples from spots of MM1 chip Add 5 ul of 50 mM HEPES to spots 1, 2 Add 5 ul of 100 mM sodium acetate pH 4 to spots 3, 4

  27. 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 ProfilingV. ProteinChipTM array Mixed Mode site (MM1) 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 Repeat process Wash chips 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

  28. 50000 100000 150000 Serum Protein ProfilingVI. Size separation Sample: Cibacron blue spin column 1 Pool K70 spin column 1M urea, 0.125% CHAPS PBS (Drain column dry, add 30 ul sample carefully to top of gel, spin 700 xg 4sec, collect filtrate, add 30 ul same buffer, spin, collect filtrate, repeat elution if necessary.) Fraction 1 2 3

  29. Serum Protein ProfilingVI. Size separation Sample: Cibacron blue spin column 2 Pool SuperDex75 spin column 1M urea, 0.125% CHAPS PBS Fraction 1 2 3 4 5 6 50000 100000 150000

  30. Serum Protein ProfilingVI. Size separation Sample: Cibacron blue spin column 2 Pool SuperDex75 spin column 1M urea, 0.125% CHAPS PBS Fraction 1 2 3 4 5 6 5000 10000 15000 20000

  31. Peptide standards (CHCA) Protein standards (SPA) At least duplicate to establish reproducibility. Recalibrate if necessary. Serum Protein ProfilingVII. Reading ProteinChipTM array 1. Check instrument performance

  32. Low laser energy (Sensitivity 6, Intensity 10-25) Average of 50 laser shots High laser energy (Neutral density filter out, Sensitivity 9, Intensity 45) Average of at least 80 laser shots At least duplicate to establish reproducibility Serum Protein ProfilingVII. Reading ProteinChipTM array 2. Read samples manually

  33. Serum Protein ProfilingVII. Reading ProteinChipTM array 3. Read samples by Automode 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 Adjustment 6: 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.

  34. 6 4 2 0 3 2 1 0 3 2 1 0 2 1.5 1 0.5 0 5000 10000 15000 Serum Protein ProfilingVIII. Representative profiles - SAX2 SPA Low laser energy Wash 1 CHCA SPA Wash 2 CHCA

  35. Serum Protein ProfilingVIII. Representative profiles - SAX2 SPA 7.5 High laser energy 5 2.5 0 Wash 1 15 CHCA 10 5 0 SPA 2 1 0 Wash 2 0.75 CHCA 0.5 0.25 0 50000 100000 150000

  36. 1 0.5 0 15 10 5 0 2 1.5 1 0.5 0 10 7.5 5 2.5 0 5000 10000 15000 5000 10000 15000 Serum Protein ProfilingVIII. Representative profiles - Ni(II) SPA Low laser energy Wash 1 CHCA SPA Wash 2 CHCA

  37. 12.5 10 7.5 5 2.5 0 6 4 2 0 3 2 1 0 4 3 2 1 0 50000 100000 150000 50000 100000 150000 Serum Protein ProfilingVIII. Representative profiles - Ni(II) High laser energy SPA Wash 1 CHCA SPA Wash 2 CHCA

  38. 7.5 5 2.5 0 SPA 6 4 Wash 1 2 CHCA 0 40 30 20 SPA 10 0 20 Wash 2 CHCA 10 0 5000 10000 15000 20000 5000 10000 15000 20000 Serum Protein ProfilingVIII. Representative profiles - H4 Low laser energy

  39. 0.4 0.3 0.2 0.1 0 0.15 0.1 0.05 0 2 1 0 0.3 0.2 0.1 0 50000 100000 150000 50000 100000 150000 Serum Protein ProfilingVIII. Representative profiles - H4 High laser energy SPA Wash 1 CHCA SPA Wash 2 CHCA

  40. 4 3 2 1 0 2 1.5 1 0.5 0 6 4 2 0 1.5 1 0.5 0 5000 10000 15000 Serum Protein ProfilingVIII. Representative profiles - MM1 (stainless steel) SPA Low laser energy Wash 1 CHCA SPA Wash 2 CHCA

  41. 0.4 0.2 0 0.75 0.5 0.25 0 1 0.5 0 0.2 0.15 0.1 0.05 0 Serum Protein ProfilingVIII. Representative profiles - MM1 (aluminum) Low laser energy 50 mM HEPES 100 mM acetate pH 4 30% ethylene glycol 10% acetonitrile 0.1% triethylamine 5000 10000 15000 5000 10000 15000

  42. 1 0.5 0 0.6 0.4 0.2 0 2 1 0 0.6 0.4 0.2 0 50000 100000 150000 50000 100000 150000 Serum Protein ProfilingVIII. Representative profiles - MM1 (stainless steel) High laser energy SPA Wash 1 CHCA SPA Wash 2 CHCA

  43. 1 0.5 0 0.4 0.2 0 1.5 1 0.5 0 0.06 0.04 0.02 0 50000 100000 150000 50000 100000 150000 Serum Protein ProfilingVIII. Representative profiles - MM1 High laser energy HEPES 5% acetonitrile 0.1% TFA 5% acetonitrile 0.1% TEA 1M urea 0.1%CHAPS HEPES/NaCl

  44. 2. Detect peaks (combine useful data from all spectra) Low mass High mass Serum Protein ProfilingIX. Data Processing 1. Arrange data of one spot into Experiment

  45. 3. Arrange peak maps of 4 spots (4 ProteinChipTM sites) into Experiment Low mass High mass 4. Combine Peak maps Serum Protein ProfilingIX. Data Processing

  46. Disease spot 1 Disease spot 2 Normal spot 1 Normal spot 2 Serum Protein ProfilingIX. Data Processing 0.05% mass error Peak count:169 Disease combine 4 2 0 10 164 Normal combine 7.5 5 2.5 0 Almost no common peaks (no red)! Disease Normal compare 2.5 0 -2.5 -5 -7.5 4000 6000 8000 10000

  47. Serum Protein ProfilingIX. Data Processing 0.15% mass error 148 135 Still too many unique peaks CONFIDENTIAL

  48. Serum Protein ProfilingIX. Data Processing 0.3% mass error 125 110 Too high mass error leading to counting 2 or more peaks as one Reasonable

  49. Serum Protein ProfilingX. PeakMaps S/N 10 0.5% mass error SAX2 Ni(II) H4 SCX1 Combine 5000 10000 15000

  50. Serum Protein ProfilingX. PeakMaps S/N 10 0.5% mass error SAX2 Ni(II) H4 AS1 Combine 150000 50000 100000

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