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DEVELOPMENT OF ESSENTIAL SAMPLE PREPARATION TECHNIQUES IN PROTEOMICS USING ULTRA-HIGH PRESSURE

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Chelli Miller
Chelli Miller

Alexander R. Ivanov HSPH Proteomics Resource Department of Genetics and Complex Diseases Harvard School of Public Health www.hsph.harvard.edu/proteomics

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DEVELOPMENT OF ESSENTIAL SAMPLE PREPARATION TECHNIQUES IN PROTEOMICS USING ULTRA-HIGH PRESSURE Alexander R. Ivanov HSPH Proteomics Resource Department of Genetics and Complex Diseases Harvard School of Public Health www.hsph.harvard.edu/proteomics
Sample Preparation: -Proteolytic Digestion ?In-solution ?In-gel ?On-membrane -Cell Lysis ?Pressure ?Organic solvents
Workflow for Optimization of Digestion Protocol Protein Mix Conventional Pressure-Assisted, PCT (In Incubator) (In Barocycler) Tested variables in optimization of digestion Dissolution Proteolytic Reduction Reduction additives: Time of enzymes: reagent/ environment: (HFIP, urea, digestion trypsin, concentration: temperature methanol) Lys-C DTT vs. TCEP and pressure LC-MS/MS analysis and database searching (Scaffold, Protein Prophet, Peptide Prophet)
Protocol Variables Digestion conditions Dissolution Protocol Additives: In Incubator (37?C) In PCT (45?C) aliquot, 12- trypsin, 40- aliquot, 1- description Shorthand 1-Lys-C, 1- 1-Lys-C, 1- trypsin, 2- additions, 2-trypsin, 2-trypsin, 2-trypsin, 1-trypsin 2-trypsin 1-trypsin protocol MeOH 40-hrs name Urea Brief HFIP 2-hr 4-hr 8-hr hrs, hrs hr hr I-A_ Standard protocol x I-C_ Standard in PCT x I-D Standard (small volume) x II-A Lys-C x II-C Lys-C in PCT x III-A HFIP (Hexafluoroisopropanol) x x III-C HFIP +PCT x x IV-A MeOH x x IV-C MeOH +PCT x x V-A Urea x x V-C Urea +PCT x x VI-A DTT for Reduction x VI-C DTT for Reduction in PCT x VII-A Urea/HFIP x x x VII-C Urea/HFIP/PCT x x x I-A-1 Standard, only (1) 12-hr tryp x I-A-2 Standard (I-A) x I-C-1 Only 1 PCT digest x I-C-2 Standard in PCT (I-C) x I-C-4 4 PCT digests x I-C-8 8 PCT digests x I-A* Standard, only (1) 12-hr tryp x I-AC* I-A, digestion in PCT x I-C* I-C, 1 tryp digest x VI-A50 VI-A with 50mM DTT x VI-C50 VI-C with 50mM DTT x
100 120 140 0 20 40 60 80 Conventional PCT Lys-C Lys-C + PCT HFIP HFIP + PCT MeOH MeOH + PCT Urea Urea + PCT Unique standard peptides 100% 120% 0% 20% 40% 60% 80% Conventional PCT Lys-C Lys-C + PCT HFIP HFIP + PCT MeOH In-Solution Tryptic Digestion, 100 fmol/analysis MeOH + PCT Urea Urea + PCT
In-Solution Tryptic Digestion: Reproducibility of Peptide Abundances
In-Solution Tryptic Digestion: Reproducibility of Peptide Abundances Average peptide abundance, PCT vs. conventional. 350000 300000 250000 200000 y = 1.207x PCT 150000 R? = 0.936 100000 50000 0 0 50000 100000 150000 200000 250000 300000 LC-MS LC-MS LC-MS Run-to-run Runs 1 and Runs 2 and Runs 1 and Mean reproducibility (R2) 2, R2 3, R2 3, R2 R2 CV Conventional Digestion 0.945 0.815 0.892 0.884 7.4% PCT Digestion 0.908 0.938 0.950 0.932 2.3% Sample-to-sample Samples 1 Samples 2 Samples 1 Mean reproducibility,(R2) and 2, R2 and 3, R2 and 3, R2 R2 CV Conventional Digestion 0.862 0.918 0.883 0.888 3.2% PCT Digestion 0.966 0.944 0.939 0.950 1.5%
In-Solution Tryptic Digestion: Differential Peptide Recovery 3 RATIO (Conv/PCT) 2 KD 1 0 -1 -2 -3
In-Solution Tryptic Digestion: Differential Peptide Recovery
In-Solution Tryptic Digestion: Differential Peptide Recovery
?Higher throughput (20x fold) ?Higher efficiency of proteolysis ?Lower preanalytical variability ?Better overall peptide recovery ?Absence of PCT-induced in vitro peptide oxidation ?Digestion specificity is not hampered by ultra-high pressure
PCT-Assisted Cell Rupture
Conventional Cell/Tissue Rupture Approaches Mechanical stress Ultrasound Osmosis Credit: R. Schlicher, R. Apkarian, and M. Baran, www.cchem.berkeley.edu, www.sciencephotolibrary.com
Pressure Cycling ¨C Assisted Lysis and Protein Extraction
Pressure Cycling-Assisted and Organic Solvent ¨C Assisted Cell Lysis
Pressure Cycling-Assisted and Organic Solvent ¨C Assisted Cell Lysis
Pressure Cycling-Assisted and Organic Solvent ¨C Assisted Cell Lysis
Pressure Cycling-Assisted and Organic Solvent ¨C Assisted Cell Lysis
GO Term Enrichment Analysis. Cellular Localization.
Conclusions (1.) higher throughput; (2.) higher efficiency; (3.) superior reproducibility of enzymatic digestion; (4.) more efficient cell lysis; (5.) superior recovery of membrane, organelle, and complex forming proteins in comparison to the conventional protocols, as well as increased identification of proteins containing TMDs.
Acknowledgements Emily Freeman Alexander Lazarev Vera Gross PBI Funding: NIEHS, HSPH GCD Department, CRDF, Harvard Catalyst

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DEVELOPMENT OF ESSENTIAL SAMPLE PREPARATION TECHNIQUES IN PROTEOMICS USING ULTRA-HIGH PRESSURE

  • 1. DEVELOPMENT OF ESSENTIAL SAMPLE PREPARATION TECHNIQUES IN PROTEOMICS USING ULTRA-HIGH PRESSURE Alexander R. Ivanov HSPH Proteomics Resource Department of Genetics and Complex Diseases Harvard School of Public Health www.hsph.harvard.edu/proteomics
  • 2. Sample Preparation: -Proteolytic Digestion ?In-solution ?In-gel ?On-membrane -Cell Lysis ?Pressure ?Organic solvents
  • 3. Workflow for Optimization of Digestion Protocol Protein Mix Conventional Pressure-Assisted, PCT (In Incubator) (In Barocycler) Tested variables in optimization of digestion Dissolution Proteolytic Reduction Reduction additives: Time of enzymes: reagent/ environment: (HFIP, urea, digestion trypsin, concentration: temperature methanol) Lys-C DTT vs. TCEP and pressure LC-MS/MS analysis and database searching (Scaffold, Protein Prophet, Peptide Prophet)
  • 4. Protocol Variables Digestion conditions Dissolution Protocol Additives: In Incubator (37?C) In PCT (45?C) aliquot, 12- trypsin, 40- aliquot, 1- description Shorthand 1-Lys-C, 1- 1-Lys-C, 1- trypsin, 2- additions, 2-trypsin, 2-trypsin, 2-trypsin, 1-trypsin 2-trypsin 1-trypsin protocol MeOH 40-hrs name Urea Brief HFIP 2-hr 4-hr 8-hr hrs, hrs hr hr I-A_ Standard protocol x I-C_ Standard in PCT x I-D Standard (small volume) x II-A Lys-C x II-C Lys-C in PCT x III-A HFIP (Hexafluoroisopropanol) x x III-C HFIP +PCT x x IV-A MeOH x x IV-C MeOH +PCT x x V-A Urea x x V-C Urea +PCT x x VI-A DTT for Reduction x VI-C DTT for Reduction in PCT x VII-A Urea/HFIP x x x VII-C Urea/HFIP/PCT x x x I-A-1 Standard, only (1) 12-hr tryp x I-A-2 Standard (I-A) x I-C-1 Only 1 PCT digest x I-C-2 Standard in PCT (I-C) x I-C-4 4 PCT digests x I-C-8 8 PCT digests x I-A* Standard, only (1) 12-hr tryp x I-AC* I-A, digestion in PCT x I-C* I-C, 1 tryp digest x VI-A50 VI-A with 50mM DTT x VI-C50 VI-C with 50mM DTT x
  • 5. 100 120 140 0 20 40 60 80 Conventional PCT Lys-C Lys-C + PCT HFIP HFIP + PCT MeOH MeOH + PCT Urea Urea + PCT Unique standard peptides 100% 120% 0% 20% 40% 60% 80% Conventional PCT Lys-C Lys-C + PCT HFIP HFIP + PCT MeOH In-Solution Tryptic Digestion, 100 fmol/analysis MeOH + PCT Urea Urea + PCT
  • 6. In-Solution Tryptic Digestion: Reproducibility of Peptide Abundances
  • 7. In-Solution Tryptic Digestion: Reproducibility of Peptide Abundances Average peptide abundance, PCT vs. conventional. 350000 300000 250000 200000 y = 1.207x PCT 150000 R? = 0.936 100000 50000 0 0 50000 100000 150000 200000 250000 300000 LC-MS LC-MS LC-MS Run-to-run Runs 1 and Runs 2 and Runs 1 and Mean reproducibility (R2) 2, R2 3, R2 3, R2 R2 CV Conventional Digestion 0.945 0.815 0.892 0.884 7.4% PCT Digestion 0.908 0.938 0.950 0.932 2.3% Sample-to-sample Samples 1 Samples 2 Samples 1 Mean reproducibility,(R2) and 2, R2 and 3, R2 and 3, R2 R2 CV Conventional Digestion 0.862 0.918 0.883 0.888 3.2% PCT Digestion 0.966 0.944 0.939 0.950 1.5%
  • 8. In-Solution Tryptic Digestion: Differential Peptide Recovery 3 RATIO (Conv/PCT) 2 KD 1 0 -1 -2 -3
  • 9. In-Solution Tryptic Digestion: Differential Peptide Recovery
  • 10. In-Solution Tryptic Digestion: Differential Peptide Recovery
  • 11. ?Higher throughput (20x fold) ?Higher efficiency of proteolysis ?Lower preanalytical variability ?Better overall peptide recovery ?Absence of PCT-induced in vitro peptide oxidation ?Digestion specificity is not hampered by ultra-high pressure
  • 13. Conventional Cell/Tissue Rupture Approaches Mechanical stress Ultrasound Osmosis Credit: R. Schlicher, R. Apkarian, and M. Baran, www.cchem.berkeley.edu, www.sciencephotolibrary.com
  • 14. Pressure Cycling ¨C Assisted Lysis and Protein Extraction
  • 15. Pressure Cycling-Assisted and Organic Solvent ¨C Assisted Cell Lysis
  • 16. Pressure Cycling-Assisted and Organic Solvent ¨C Assisted Cell Lysis
  • 17. Pressure Cycling-Assisted and Organic Solvent ¨C Assisted Cell Lysis
  • 18. Pressure Cycling-Assisted and Organic Solvent ¨C Assisted Cell Lysis
  • 19. GO Term Enrichment Analysis. Cellular Localization.
  • 20. Conclusions (1.) higher throughput; (2.) higher efficiency; (3.) superior reproducibility of enzymatic digestion; (4.) more efficient cell lysis; (5.) superior recovery of membrane, organelle, and complex forming proteins in comparison to the conventional protocols, as well as increased identification of proteins containing TMDs.
  • 21. Acknowledgements Emily Freeman Alexander Lazarev Vera Gross PBI Funding: NIEHS, HSPH GCD Department, CRDF, Harvard Catalyst
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