1. ABSTRACT Two-dimensional electrophoretic (2-DE) analysis of differential protein expression is limited by the number of proteins detectable on a single gel. One technological area that directly addresses this limitation is improved sample preparation and protein solubilization. This presentation describes the application of various sample preparation techniques aimed at increasing the protein analytical power of 2-DE. Pressure Cycling Technology (PCT) uses alternating cycles of high and low pressures to induce cell lysis. Tissues placed in specially designed PULSE  Tubes were subjected to alternating cycles of maximum (35,000 PSI) and minimum (ambient) pressures in the Barocycler  [1]. Mouse liver lysates produced by sonication or with a ground glass tissue grinder were compared to lysates produced by PCT. PDQuest analysis of two-dimensional gels of the PCT lysate revealed 2,126 protein spots compared to 1,832 protein spots in duplicate gels of the tissue grinder lysate. The lysate produced by sonication yielded 1,739 protein spots with a preponderance towards diminished recovery of high molecular weight proteins. This corresponded with a general increase in the spot intensities of low molecular weight proteins suggesting that some protein degradation might be occurring during sonication. (Supported in part by AFOSR Grant F49620-03-1-0089 (FAW)) METHODS 250 mg of fresh, ice-cold saline-perfused mouse and rat liver samples were excised and frozen. Ground-glass (GG) Homogenization Eight (8) volumes (2 mL) of lysis buffer (9M urea, 4% NP-40, 0.5% ampholytes pH 3-10, 1% DTT) were added to the samples in a 50 mL beaker; the samples were thoroughly minced with surgical scissors [2]. Minced liver was transferred to GG tubes for homogenization (at RT). Lysates were stored in microcentrifuge tubes until 2-DE. Sonication (Son) Eight volumes (2 mL) of lysis buffer (9M urea, 4% NP-40, 1% ampholytes pH 3-10, 1% DTT) were added to the liver sample and sonicated. Pressure Cycling Technology (PCT) Mouse liver (250 mg) or rat liver samples (250 mg) were placed in specially designed PULSE Tubes (Figure 1) with 1.25 mL of the lysis buffer described above and subjected to alternating cycles of high and low pressures in a pressure-generating instrument (Barocycler Model NEP3229, Figure 2). PULSE Tubes were subjected to 10 pressure cycles; each cycle consisted of 20 seconds at 35,000 PSI followed by 20 seconds at ambient pressure. Following PCT, the lysates were collected and cellular debris was removed by centrifugation at 20,000 RCF for 10 minutes. 2-DE Protein concentration was determined using an Amido black assay 1st dimension = pH 3-10 non-linear, BioRad 24 cm IPG strips, 500 μg loaded onto each strip by passive rehydration 2nd dimension = 11-17% polyacrylamide, 20 x 25 x 0.15 cm Gels were run in a 20 gel DALT tank to minimize variation Gels were stained with colloidal CBB [3], scanned, and analyzed with PDQuest to detect the number of spots for each sample RESULTS In the initial experiment, replicate mouse liver samples (n=2) were solubilized as described. PDQuest detected the following in each preparation group (see Figure 3): PCT2,126 ± 44 Sonication1,739 ± 178 Ground glass1,832 ± 21 PCT significantly improved protein spot detection over both sonication (22%) and ground glass homogenization (16%). Sonication and GG results did not differ significantly. Many higher molecular weight proteins were significantly diminished in the sonicated sample (see Figure 4) compared to PCT and GG. A second experiment compared rat liver sample (n=5) preparation by PCT, GG, GG with sonication, and GG/sonication in twice the lysis buffer volume (16 volumes). PDQuest detected the following in each preparation group (see Figure 5): PCT 2,280 ± 173 GG 1,620 ± 137 GG/Son 1,735 ± 144 GG/Son/2X 1,682 ± 165 PCT significantly improved protein spot detection over GG (40%), GG/Son (31%), and GG/Son/2X (36%). None of the GG-based preparation methods differed significantly from one another. Representative gel images of each of these groups are shown in Figure 6. INTRODUCTION Two-dimensional electrophoresis (2-DE) is a powerful protein analytical tool whose major strengths include semi-global quantitation and charge separation of complex protein mixtures, enabling the analysis of variable post-translational modification. One of 2-DE’s limitations relates to its limited dynamic range and consequently the number of proteins expressed that can be analyzed on a single gel. In an attempt to improve the yield of detectable proteins during sample preparation, we applied a novel extraction technique, Pressure Cycling Technology (PCT). PCT uses alternating cycles of high and low pressures to induce cell lysis. Our hypothesis contends that tissue samples subjected to PCT in the presence of a suitable lysis buffer will liberate a greater abundance of cellular proteins for subsequent 2-DE and improve the utility of this proteomic approach. Application of Pressure Cycling Technology (PCT) in Proteomics: Increased Yield of High Molecular Weight Proteins in Mouse Liver Lysates Frank Witzmann 1, Heather Ringham 1, Gary Smejkal 2, James Behnke 2 1 Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN, USA 2 Pressure BioSciences, Inc., West Bridgewater, MA, USA CONCLUSIONS This study confirms and extends previous results with E. coli where PCT sample preparation for 2-DE improved protein spot detection by 14.2% compared to standard bead mill treatment [4]. In mouse liver samples, PCT improved overall protein extraction from tissue as indicated by a significantly higher number of protein spots detected by 2-DE. In rat liver samples, PCT demonstrated an even greater improvement in protein extraction. To overcome some of 2-DE’s limitations (with respect to sensitivity), it is beneficial to include PCT whole tissue sample preparation protocols. To better understand the mechanism of PCT’s utility, ongoing experiments are aimed at identifying proteins where relative abundance on 2-DE is selectively enhanced (or decreased) by PCT in lysis buffer. Additional studies are necessary to optimize the PCT conditions (duration, number of cycles, etc.) for unique tissue and cell culture samples. Figure 1. PULSE Tubes (PT). Liver samples were placed on the lysis disk in the PT and subjected to 10 pressure cycles in the Barocycler instrument. The combination of physical passage through the PT lysis disk, rapid pressure changes, and other bio-physical mechanisms effectively induced cell lysis. Figure 2. The Barocycler NEP3229 bench top instrument used to extract liver samples in this study is capable of processing up to three samples simultaneously. Sonication PCT Ground Glass calreticulin grp78 hsc70 PDI F1 ATPase beta endoplasmin 100 80 60 50 40 MW (kDa) Figure 4. Sections of representative mouse liver (500 μg) gel images (pI range ~4.5-6.5 and MW ~30-100 kDa) illustrating the loss of higher MW proteins with sonication. Overall protein spot detection is significantly higher in PCT prepared samples. F= 6.167 P < 0.005 n = 5 Spots Detected 0 500 1000 1500 2000 2500 PCTGG/SonGGGG/Son Volx2 ComparisonP valueSignificant? PCT vs. GG/No Son 0.002 Yes PCT vs. GG/Son/x2 Vol 0.003 Yes PCT vs. GG/Son 0.006 Yes GG/Son vs. GG/No Son 0.519 No GG/Son/x2 Vol vs. GG/No Son 0.728 No GG/Son vs. GG/Son/x2 Vol 0.764 No * * * Rat Liver Sample Preparation Figure 5 Mouse Liver Sample Preparation 0 500 1000 1500 2000 2500 Son PCT GG Spots Detected ComparisonP valueSignificant? PCT vs. Son 0.03 Yes PCT vs. GG 0.02 Yes Son vs. GG 0.816 No t-test n = 2 Figure 3. * * PCT GG/Son GG GG/Son vol 2X Figure 6. Representative rat liver 2D gel images (pI range 3-10 and MW ~5-150 kDa) illustrating colloidal Coomassie blue-stained rat liver samples (500 μg) prepared as labeled. Overall protein spot detection is significantly higher in PCT prepared samples. REFERENCES 1.Schumacher R.T., M. Manak, P. Garrett, W. Miller, N. Lawrence, and F. Tao. (2002). Automated Solution for Sample Preparation: Nucleic Acid and Protein Extraction From Cells and Tissues Using Pressure Cycling Technology (PCT). Am. Laboratory 34 (16): 38-43. 2.Witzmann, F.A. (2005). Preparation of Mammalian Tissue Samples for Two- dimensional Electrophoresis. In: The Proteomics Protocols Handbook, J.M. Walker, ed., The Humana Press, Totowa, NJ, pp. 31-35. 3.Candiano G., M. Bruschi, L. Musante, L. Santucci, G.M. Ghiggeri, B. Carnemolla, P. Orecchia, L. Zardi & P.G. Righetti. (2004). Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327-1333. 4.Smejkal, G.B. M.H. Robinson, N.P. Lawrence, F. Tao, C.A. Saravis, and R.T. Schumacher. (2006). Increased Protein Yields From Escherichia coli Using Pressure Cycling Technology. J. Biomolecular Techniques (in press).