Digestion with Pressure Cycling Technology (PCT)
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1 Digestion with Pressure Cycling Technology (PCT) Nicholas Sobol, Jennifer Oprihory*, Tiffany Remsen, A. Yuen, P. Pevsner Department of Pathology and Anatomical Sciences University of Missouri School of Medicine Columbia, MO
2 What is PCT? PCT is the application of cycling high and low hydrostatic pressures to a solid (tissue) or liquid (trypsin digest) sample. Suzuki C, Suzuki K. J Biochem 1962; 52: 67. Ruan K, Lange R, Bec N, Balny C. Biochem Biophys Res Commun 1997; 239: 150. Chicon R, Belloque J, Recio I, Lopez-Fandino R. J Dairy Res 2006; 73: 121. Smejkal GB, Robinson MH, Lawrence NP, Tao F, Saravis CA, Schumacher RT. J Biomol Tech 2006;17: 173. Ringham H, Bell RL, Smejkal GB, Behnke J, Witzmann FA. Electrophoresis 2007; 28: Pevsner P, Vecchione D, Stall B, Remsen T, Anand S, Stern A British Mass Spectrometry Society, Edinburgh, Scotland, Pevsner P, Vecchione D, Remsen T, Kessler P, Levers N, Yang P, Stern A, Samuels H , British Mass Spectrometry Society, 2007 Robinson College,Cambridge. Pevsner P, Vecchione D, 1 Remsen T, Kessler P, Momeni M, Duddempudi S, Francois F, Stern A, Anand S British Mass Spectrometry LCMS Meeting, Robinson College, Cambridge, UK,
3 PCT Sample Assembly Microtubes (150µl) Cartridge Stack (12-48) Microtube Cartridge (6 or 8 tubes)
4 PCT Barocycler
5 How does PCT Work? Pressures between 29k-145k PSI (2-10 kbar) denature protein Water-water and protein-water hydrogen bonds increase. Protein-protein hydrogen bonds decrease. Silva JL, Weber G. Annu Rev Phys Chem 1993; 44: 89.
6 How does PCT Work? Hydrophobic groups exposed to water. Water penetrates protein, increasing internal pressure. Hydrogen bonds responsible for secondary and tertiary structure of protein destabilize. Ruan K, Lange R, Bec N, Balny C. Biochem Biophys Res Commun 1997; 239: 150.
7 How does PCT Work? Non-native salt bridges, hydrophobic, and electrostatic interactions disrupted Electrostatic replusions increase. Repulsions favor unfolding. Balny C. Journal of Physics: Condensed Matter 2004; 16: S1245.
8 PCT and Trypsin Digest Pressures >35k PSI(2.4 kbar) will denature trypsin. Pevsner P, Vecchione D, Stall B, Remsen T, Anand S, Stern A British Mass Spectrometry Society, Edinburgh, Scotland Pevsner P, Vecchione D, Remsen T, Kessler P, Levers N, Yang P, Stern A, Samuels H. British Mass Spectrometry Society, 2007 Robinson College, Cambridge. Pevsner P, Vecchione D, 1 Remsen T, Kessler P, Momeni M, Duddempudi S, Francois F, Stern A, Anand S British Mass Spectrometry LCMS Meeting, Robinson College, Cambridge, UK Lopez-Ferrer D, Petritis K, Hixson KK, Heibeck TH, Moore RJ, Belov ME, Camp DG, Smith RD. J Proteome Res 2008; 7: 3276.
9 PCT Applications Direct protein extraction from tissue Trypsin digest
10 Example of PCT Protein Extraction from Tissue
11 Extraction of Protein from Irradiated Mouse Heart Control (Normal Mouse Heart) Experimental [Mouse Heart 6 days post-total body irradiation (TBI)] -1 Gy, 2 Gy, and 4 Gy samples All samples run in triplicate Bradford Protein Assay
12 PCT Mouse Heart Protein Extraction tube sample tissue mass protein assay extraction mg mg/ml efficiency (%) HRT Na control HRT Nb HRT Nc HRT 1-6a 1 Gy HRT 1-6b HRT 1-6c HRT 2-6a 2 Gy HRT 2-6b HRT 2-6c HRT 4-6a 4 Gy HRT 4-6b HRT 4-6c ± 2.6
13 Why PCT Extraction? Reproducibility Speed Frozen samples can be processed Virtually adiabatic Ambient environment Instrument reliability 30k cycles before our first repair
14 PCT Trypsin Digest
15 PCT Trypsin Digest ( ) Remsen, T, Kessler, P, Francois, F, Stern, A, Anand, S, & Pevsner, P (2008). Imaging MALDI of colorectal carcinoma - field defects in satellite tissue. American Chemical Society Mid-Atlantic meeting, Queensborough College, Queens, NY, May, American Chemical Society Mid-Atlantic Meeting, Queensborough College, Queens, NY. Remsen, T, Naftolin, F, Talebian, S, Kessler, P, Licciardi, F, Grifo, J, et al. (2008). LCMS identification of competent embryo biomarkers. British Mass Spectrometry Society, York, UK, September, Lopez-Ferrer D, Petritis K, Hixson KK, Heibeck TH, Moore RJ, Belov ME, Camp DG,2nd, Smith RD. J Proteome Res 2008; 7: Pevsner PH, Melamed J, Remsen T, Kogus A, Francois F, Kessler P, Stern A, Anand S. Biomarkers Med 2009; 3: 55.
16 Trypsin/ Calibrant Prep Trypsin Trypsin was dissolved in 100 mm ammonium bicarbonate Calibrant Calibrants were dissolved in 0.1% TFA/Acetonitrile (ACN), 30/70.
17 BSA* BSA/Cyto-C Prep 1158 µg BSA / 2 µg trypsin in 160 µl of 100 mm ammonium bicarbonate Cytochrome C* 1158 µg Cyto-C / 2 µg trypsin in 160 µl of 100 mm ammonium bicarbonate *No alkylation or reduction was performed on any samples.
18 PCT Protocol Experimental Protocol 35 kpsi (2.4 kbar) 40 seconds/cycle (with compressor) Cycle= 20 sec 35k PSI / 20 sec 1 atm (5 seconds with continuous N 2 gas supply) BSA (16) and Cyto-C (16) 1 cycle (n=4) 5 cycles (n=4) 10 cycles (n=4) 20 cycles (n=4)
19 Reference Sample Protocol BSA (4) & Cyto-C (4) 30 Minutes, 55 C (n=1) 1 Hour, 55 C (n=1) 2 Hours, 55 C (n=1) 4 Hours, 55 C (n=1)
20 Matrix 70% ACN/ 30% CHCA 5 mg/ml
21 MALDI Instrument Axima TOF 2 (MALDI TOF-TOF Shimadzu, Kyoto, Japan) Reflectron mode
22 Results MASCOT Identification of Cytochrome C & BSA Numbered scores represent exact matches. Coverage maps indicate % coverage and matching peptide sequences.
23 PCT Trypsin Digest Spectra
24 BSA 1 Cycle
25 BSA 1 Cycle Score
26 BSA 1 Cycle Coverage Map
27 BSA 5 Cycles
28 BSA 5 Cycles Score
29 BSA 5 Cycles Coverage Map
30 BSA 10 Cycles
31 BSA 10 Cycles Score
32 BSA 10 Cycles - Coverage Map
33 BSA 20 Cycles
34 BSA 20 Cycles Score
35 BSA 20 Cycles - Coverage Map
36 Cyto-C 5 Cycles
37 Cyto-C 5 Cycles Score
38 Cyto-C 5 Cycles -Coverage Map
39 Cyto-C 10 Cycles
40 Cyto-C 10 Cycles
41 Cyto-C 10 Cycles Coverage Map
42 Cyto-C 20 Cycles
43 Cyto-C 20 Cycles Score
44 Cyto-C 20 Cycles Coverage Map
45 Reference Sample Spectra
46 Cyto-C 4 Hours 55 C C
47 Cyto-C 4 Hours 55 C Score C
48 Cyto-C 4 Hours 55 C - Coverage Map
49 BSA 2 Hours 55 C
50 BSA 2 Hours 55 C Score
51 BSA 2 Hours 55 C Coverage Map
52 BSA (Bovine Serum Albumin) + Trypsin + PCT (Samples 1-8) 158 µl BSA (1158 µg) + 2 µl trypsin (2 µg); BSA: Trypsin, 579:1 Runs (Cycles/Seconds) MASCOT Score Sequence Coverage (%) 1/ /20 No match N/A 1 /20 No match N/A 1/20 No match N/A 5/ /100 No match N/A 5/100 No match N/A 5/100 No match N/A
53 BSA (Bovine Serum Albumin) + Trypsin + PCT (Samples 8-16) 158 µl BSA (1158 µg) + 2 µl trypsin (2 µg); BSA: Trypsin, 579:1 Runs (Cycles/Seconds) MASCOT Score Sequence Coverage (%) 10/ / /200 No match N/A 10/200 No match N/A 20/ / / /400 No match N/A
54 Cyto-C (Horse Heart Cytochrome-C) + Trypsin + PCT (Samples 1-8) 158 µl Cyto-C (1158 µg) + 2 µl trypsin (2 µg); Cyto-C: Trypsin, 579:1 Runs (Cycles/Seconds) MASCOT Score Sequence Coverage (%) 1/20 No match N/A 1/20 No match N/A 1/20 No match N/A 1/20 No match N/A 5/ / / /100 No match N/A
55 Cyto-C (Horse Heart Cytochrome-C) + Trypsin + PCT (Samples 8-16) 158 µl Cyto-C (1158 µg) + 2 µl trypsin (2 µg); Cyto-C: Trypsin, 579:1 Runs (Cycles/Seconds) MASCOT Score Sequence Coverage (%) 10/ / / / / / / /
56 BSA Reference 55 C Time Mascot Score Sequence Coverage (%) 30 Minutes No match N/A 1 Hour Hours Hours No match N/A
57 Cytochrome C Reference 55 C Time Mascot Score Sequence Coverage (%) 30 Minutes No match N/A 1 Hour No match N/A 2 Hours No match N/A 4 Hours 99 78
58 Data Summary Protein identification is cycledependent. PCT digest in minutes achieves equal coverage compared to water bath digest (55 C) at 2 and 4 hours, respectively, for BSA and Cytochrome C.
59 BSA Tertiary Structure Disulfide Bond Effects on Trypsin Digest 34 cysteine residues 17 disulfide bonds Cytochrome C 2 cysteine residues (CAQC- no disulfide bond) P. Restani, C. Ballabio, A. Cattaneo, et. al. Allergy 2004; 59: 21.
60 Why PCT Trypsin Digest? Reduced digest time (hrs to mins) Ambient temperature digest Reduced analyte / trypsin ratio (~500:1) No tissue maceration
61 Conclusion PCT tissue extraction is: 1) rapid 2) reproducible 3) high-yield PCT trypsin digest is: 1) rapid with high % coverage 2) reproducible
62 Pevsner Laboratory Back Row (L to R): Alvis Yuen, Nick Sobol, Daniel Weissman,Jennifer-Leigh Oprihory, Ariel Gross Middle Row: Tiffany Remsen, Arko Mukherjee, Robert Chouake Front Row: Michael Yee & Paul Pevsner
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