Application of Pressure Cycling Technology to the Proteomic Analysis of Rat Liver 1University

Douglas Hinerfeld1, Sunny Tam1 & Gary Smejkal2 of Massachusetts Proteomics Consortium, Worcester, MA, 2Pressure BioSciences, West Bridgewater, MA

Abstract: Comprehensive proteomic analysis of mammalian tissues requires a reproducible and efficient means by which proteins can be extracted. The use of a mortar and pestle in the presence of liquid nitrogen, followed by further homogenization with a Polytron, results in effective solublization of solid tissues however, the process is laborious and potentially dangerous if dealing with infectious samples. We have tested an alternative method of extraction (Pressure Cycling Technology, or PCT), using rat liver protein. PCT uses alternating cycles of high and low pressure to induce cell lysis in a fully programmable, pressure generating instrument (Barocycler™). Tissues are placed in specially designed, single-use PULSE™ Tubes, which are then placed inside the Barocycler instrument and subjected to multiple pressure cycles with maximum pressure reaching 35,000 PSI. Protein from rat liver was extracted using the standard mortar and pestle/Polytron method and was compared to PCT by protein assay and 2D gel analysis. The results show a highly comparable proteomic profile between the solublization methods with few distinct differences that have been characterized by Mass Spectrometry. Methods: •Mortar and Pestle/Polytron: Frozen pieces of rat liver were pulverized with a mortar and pestle in liquid nitrogen and transferred to a centrifuge tube. Three milliliters COMS extraction buffer (7M urea, 2M thiourea, 1% C7, 40mM Tris) was added, and samples were homogenized with a polytron.

By repeated cycling of high (35,000 psi) and low pressures in combination with shearing through the lysis disk in a PULSE rube in a denaturing buffer, proteins are extracted for proteomic analysis

Table 1: Comparison of protein yields from different extraction methods Extraction Method Mortar and Pestle/Barocycler

Whole Tissue/Barocycler

•Mortar and Pestle/Barocycler: Frozen pieces of tissue were pulverized with a mortar and pestle in liquid nitrogen and transferred to a PULSE tube. COMS buffer was added to a final volume of 1.25mls, and the samples were homogenized by pressure cycling under the following parameters; 35,000 psi for 20 sec., ambient pressure for 20 sec. Repeated 10 times. •Whole tissue/Barocyler: Whole pieces of rat tissue were transferred to a PULSE tube. COMS buffer was added to a final volume of 1.25mls, and the samples were homogenized by pressure cycling under the following parameters; 35,000 psi for 20 sec., ambient pressure for 20 sec. Repeated 10 times. Once homogenized all samples were reduced with tri-butlyphosphine and alkylated with acrylamide for 90 min and then subjected to centrifugation at 57,000xG for 20 min to remove insoluble protein. Soluble protein was precipitated with 9 volumes acetone, precipitated protein was solublized in resuspension solution (7M urea, 2M thiourea, 2%CHAPS), and protein concentrations were determined by Bradford assay. 100µg protein was subjected to two-dimensional electrophoresis on GE 11cm pH 3-10 IPG strips and Bio-Rad 8-16% criterion gels in duplicate. Gels were stained with Sypro Ruby and imaged on a Bio-Rad Gel Doc. Image analysis was performed on Nonlinear Progenesis Discovery software, and spots of interest were excised on a Bio-Rad EXQuest robotic spot cutter. Proteins were subjected to in-gel digests with trypsin, desalted with C18 Zip-tips and subjected to MALDI MS/MS analysis on a Kratos QIT or LC MS/MS on a Thermo-Finnegan LTQ.

Figure 3: Differentially extracted proteins

Figure 1: Pressure cycling technology

Mortar and Pestle/Polytron

mg tissue/ml lysis buffer 123 106 142 107 117 95 106 58 66

mg protein/mg frozen tissue 0.135 0.173 0.154 0.172 0.143 0.171 0.147 0.178 0.217

1, 2, 3 = Mortar and Pestle/Barocycler 4, 5, 6 = Whole tissue/Barocycler 7, 8, 9 = Mortar and Pestle/Polytron 2D gel image analysis was performed to identify proteins that were differentially present between the extraction methods. Each bar represents the average of the spot volume across duplicate gels.

Comparable protein yields are achieved by the three protein extraction methods. For all methods, the greater the volume of lysis buffer to mg of tissue, the better the extraction efficiency

Figure 2: Reproducibility of protein extraction methods Mortar/Pestle Barocycler Whole Tissue Barocycler Mortar/Pestle Polytron Each extraction method was repeated in triplicate. A representative pI 3-10 2D gel from each extraction method is shown, demonstrating a high degree of reproducibility

Table 2: MS/MS Identification of differentially extracted proteins Spot # 262 508 906 921 943 967 1067 1505 1552 1798

Protein ID TUMOR REJECTION ANTIGEN GP96 TREMBL:Q7TP27 methylcrotonoyl-Coenzyme A carboxylase 2 (Beta) Estrogen sulfotransferase Estrogen sulfotransferase ATP synthase, alpha Quinone Reductase Aldolase B DIHYDROPTERIDINE REDUCTASE FATTY ACID-BINDING PROTEIN ELONGATION FACTOR 2 Tumor rejection antigen gp96, and hnRNP U protein

Summary: Proteomic analysis of tissue requires an efficient, reproducible and safe method of protein extraction. Pressure cycling provides an effective means for extracting protein from solid tissue for proteomic analysis. The process of pulverizing the frozen tissue with the mortar and pestle, while effective, increases the labor and exposure to potentially infective agents. This data demonstrates that it is not necessary to manipulate the tissue prior to pressure cycling to achieve efficient and reproducible protein extraction. The image analysis of the 2D gels reveals distinct protein spots that are differentially present under the various methods. The identity of these proteins shows no clear pattern that suggests that the different extraction methods have no particular bias towards or against any class of proteins. Using the bench-top Barocyler, three samples can be processed simultaneously in about 7 minutes, although it is likely that the time can be significantly reduced for liver. It is possible that for more fibrous tissues such as muscle, pulverization of the tissue prior to pressure cycling may provide some benefit.