DEVELOPMENT OF ESSENTIAL SAMPLE
   PREPARATION TECHNIQUES IN
  PROTEOMICS USING ULTRA-HIGH
            PRESSURE
          ...
Sample Preparation:

-Proteolytic Digestion
   In-solution
   In-gel
   On-membrane


-Cell Lysis
   Pressure
   Orga...
Workflow for Optimization of Digestion Protocol
                                       Protein Mix

     Conventional     ...
Protocol Variables
                                                                                        Digestion condi...
100
                                                        120
                                                          ...
In-Solution Tryptic Digestion: Reproducibility of
              Peptide Abundances
In-Solution Tryptic Digestion: Reproducibility of
                      Peptide Abundances
                               ...
In-Solution Tryptic Digestion: Differential Peptide
                          Recovery
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 r...
PCT-Assisted Cell Rupture
Conventional Cell/Tissue Rupture Approaches




Mechanical stress                          Ultrasound                     ...
Pressure Cycling – Assisted Lysis and Protein Extraction
Pressure Cycling-Assisted and Organic Solvent –
               Assisted Cell Lysis
Pressure Cycling-Assisted and Organic Solvent –
               Assisted Cell Lysis
Pressure Cycling-Assisted and Organic Solvent –
               Assisted Cell Lysis
Pressure Cycling-Assisted and Organic Solvent – Assisted
                        Cell Lysis
GO Term Enrichment Analysis. Cellular Localization.
Conclusions




(1.) higher throughput;
(2.) higher efficiency;
(3.) superior reproducibility of enzymatic digestion;
(4.)...
Acknowledgements


       Emily Freeman
     Alexander Lazarev
        Vera Gross
            PBI




         Funding:
NI...
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DEVELOPMENT OF ESSENTIAL SAMPLE PREPARATION TECHNIQUES IN PROTEOMICS USING ULTRA-HIGH PRESSURE

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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

  1. 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. 2. Sample Preparation: -Proteolytic Digestion In-solution In-gel On-membrane -Cell Lysis Pressure Organic solvents
  3. 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. 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. 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. 6. In-Solution Tryptic Digestion: Reproducibility of Peptide Abundances
  7. 7. In-Solution Tryptic Digestion: Reproducibility of Peptide Abundances Average peptide abundance, PCT vs. conventional. 350000 300000 250000 200000 y = 1.207x PCT 150000 R² = 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. 8. In-Solution Tryptic Digestion: Differential Peptide Recovery 3 RATIO (Conv/PCT) 2 KD 1 0 -1 -2 -3
  9. 9. In-Solution Tryptic Digestion: Differential Peptide Recovery
  10. 10. In-Solution Tryptic Digestion: Differential Peptide Recovery
  11. 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
  12. 12. PCT-Assisted Cell Rupture
  13. 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. 14. Pressure Cycling – Assisted Lysis and Protein Extraction
  15. 15. Pressure Cycling-Assisted and Organic Solvent – Assisted Cell Lysis
  16. 16. Pressure Cycling-Assisted and Organic Solvent – Assisted Cell Lysis
  17. 17. Pressure Cycling-Assisted and Organic Solvent – Assisted Cell Lysis
  18. 18. Pressure Cycling-Assisted and Organic Solvent – Assisted Cell Lysis
  19. 19. GO Term Enrichment Analysis. Cellular Localization.
  20. 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. 21. Acknowledgements Emily Freeman Alexander Lazarev Vera Gross PBI Funding: NIEHS, HSPH GCD Department, CRDF, Harvard Catalyst

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