Improved Protein Extraction and Identification from Archival Formalin-fixed Paraffin-embedded Human Aortas using High Pressure and Heat
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1 Improved Protein Extraction and Identification from Archival Formalin-fixed Paraffin-embedded Human Aortas using High Pressure and Heat Zongming Fu & Jennifer Van Eyk Johns Hopkins University United States HUPO Meeting March 11,2013
2 Relationship of Atherosclerosis in Young Men to Serum Lipoprotein Cholesterol Concentrations and Smoking Cohort: The Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Age Range Sample #s JAMA. 1990, Dec.; 264(23)
3 Study Objectives To optimize PROTEIN EXTRACTION from FFPE Aorta for biomarker discovery To evaluate QUALITY of PDAY aorta samples Sample Type Sample # unfixed 3 FF 3 FFPE 2 Harvest condition Fresh Frozen (-80ºC) Storage Time Formalin-fixed 3 15 years Formalin-fixed, paraffin-embedded Origin - 3 months - 2 years Johns Hopkins University Hospital 3 months 15 years PDAY
4 Aorta Tissue (60 mg WT) FF FFPE Fresh Frozen 15 year storage Atmospheric Pressure: 25 o C Subdivide Tissue Dounce- Homogenize Different Conditions Atmospheric Pressure: 1 hour at 95 o C Trypsin Digest Protein Precipitation Supernatant Isolation Barocycler: 1 hour at 95 o C under 40,000 psi Sample Desalting Mass Spectrometry Data Dependent LC-MS/MS Targeted Quantitation (MRM)
5 Pressure Generation Charge pump EOS sensor Intensifier Charge pump Check valve Air Press Transducer Directional Valve Press Regulator Closure with vent & CV Cover Safety Switch.. Sample Chamber Charge Pump (150psi/ 120V or 230V/ 45W) Controller Pressure Media Fill Bottle.. End of Stroke Sense Switch Pressure Switch.. HP Check Valve LED Temperature Display LED Pressure Display Customer air supply (>85psi) Pressure Regulator ER 1 Valve Command Pressure Command Signal 4 Way Valve Muffler Extend Intensifier Extend Intensifier Retract P Pressure Transducer PBI Air-Water Intensifier Air (85psi min.) High pressure water (35ksi max.) Low pressure water (150psi max.) Electri Electric c
6 Experimental Impact FFPE Sample Preparation Conditions PRESSURE and HEAT improve Extraction Yield *All proteins were extracted from 60 mg of wet tissues except the FFPE aortas were from 20, and 31mg and normalized to 60 mg for comparison.
7 Experimental Impact FFPE Sample Preparation Conditions Room Temperature Room Temperature # Protein Identifications Heat Heat+Pressure Heat Heat+Pressure Heat Heat+Pressure Unfixed FF (>3 mo) FF (>15 years) FFPE (>15 years) Mascot IDs Sequest IDs Room Temperature Room Temperature % Identified Spectra: Sequest Heat Heat+Pressure Heat Heat+Pressure Heat Heat+Pressure Unfixed FF (>3 mo) FF (>15 years) FFPE (>15 years) % Identified Spectra
8 Experimental Impact FFPE Sample Preparation Conditions PRESSURE improves protein identification number and yield more spectra for some proteins in FFPE samples Replicate 1: Replicate 2: Heat Only Heat and Heat + Pressure Heat + Pressure Only More spectra: Heat + Pressure
9 Experimental Impact FFPE Sample Preparation Conditions PRESSURE and HEAT improve Sequence Coverage % Sequence Coverage # Unique Peptides Heat Only Heat + Pressure Fibronectin Filament A 0 Fibronectin Filament A
10 Quantitative SRM Assay Periostin Area A) B) Blue: 587.8/841.5 I.S.: 591.7/849.5 Area 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 Time (min) 0.0e Area Blue: 587.8/205.0 I.S.: 591.7/205.0 Concentration (fmol/μl) C) S/N In Buffer * In Aorta Extract * LLOD pg pg LLOQ pg pg *Amount of periostin on column D) Time (min) No. Peptides Transition Ion R.T. (min) CE 1 GFEPGVTNILK / b / y / y IIDGVPVEITEK / y / y / y10 29
11 Conclusions High PRESSURE and HEAT improves total protein extraction and identification from FF and FFPE aortic tissue FFPE tissue is better preserved than FF long term
12 Acknowledgements Johns Hopkins University Dr. Jennifer Van Eyk Dr. Allen Everett Dr. Karen Yan Mr. Avi Rosenburg Dr. Zhicheng Jin Dr. Barbara Crain Wake Forest University David Herrington Louisiana State University Dr.Grace Athas Dr. Richard S. Vander Heide Funding: NHLBI Vendor: Pressure Bioscience Ms. Roxana McCloskey Mr. Jim Behnke
13 New Model of PBI Instrument HUB 440
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