Introduction
Efficient, reproducible and rapid tissue disruption and extraction of biomolecules are prerequisite for many biological applications. Solid tissues, especially tough or fibrous ones like muscle, generally require extensive mechanical disruption prior to extraction. Mortar and pestle grinding, pulverization in liquid nitrogen or homogenization with a dounce or polytron homogenizer are some of the classical methods that have been used for tissue disruption. However, these manual methods are often inconsistent, time consuming and potentially hazardous. In addition, due to the amount of sample loss inherent in these methods, they are often unsuitable for use with small samples in the 10 μg size range. Here we describe a system for efficient tissue disruption and extraction of protein or RNA from solid tissues using the Pressure Cycling Technology Sample Preparation System (PCT SPS) and the FT 500-ND PULSE Tubes. Pressurization of small sample volumes in these tubes causes repeated compression of the sample tissue between the PULSE Tube Cap and Ram. This high-pressure mechanical tissue disruption, combined with the power of pressure cycling technology (PCT), is an efficient and reproducible method to prepare whole tissue lysates from solid tissue samples for extraction of proteins or nucleic acids.

Rhodopseudomonas palustris is a Gram negative, purple, non-sulfur, phototropic bacterium, and is a metabolically versatile microbe. The bacterium can grow in the presence or absence of oxygen. In response to environmental changes, it can engage in alternative metabolic processes for cellular respiration. R. palustris can degrade the aromatic compounds comprising lignin, the second most abundant natural polymer. As such, it is being investigated for its potential in the removal of environmental pollutants [1]. The genome of R. palustris has been sequenced and annotated [2]. It follows that the analysis of this microorganism’s proteome has become an active area of research. Reliable proteomic analysis is contingent on the efficiency by which cells are lysed and their protein constituents released. Standard technique to efficiently lyse Gram-negative bacteria requires mechanical disruption of the cell, and either enzymatic or chemical breakdown of the cell wall.

Extraction of proteins from extensively calcified osseous tissue, such as cortical bone has been particularly challenging for traditional methods of sample preparation. However, a comprehensive proteomic analysis of bone is only possible when the total protein constituency is effectively isolated. The efficiency of sample preparation is therefore a critical component of the analytical process. Historically, extraction of protein from bone required prolonged acid demineralization over several days to enable complete penetration of histochemical reagents to cellular components. Here we describe a method for the extraction of protein from ostrich tibia, which was used as a model sample to develop an extraction process that uses pressure cycling technology (PCT) and also which obviates the need for acid demineralization prior to extraction. The ability to extract proteins from bone without prior demineralization offers important advantages in efficient representative extraction of protein and significant time savings during sample preparation.

Introduction
The mass extinction of the dinosaurs, marked by the Cretaceous-Tertiary boundary, pales in magnitude compared to other lesser known mass extinction events, such as the Permian-Triassic boundary. As over 99% of all of the species that ever lived are now extinct, our understanding of biological processes has been limited by what we have learned from the fewer than 1% of species that have survived more than five major mass extinction events. Recently, collagen peptides were reportedly recovered from mineralized skeletal elements of Tyrannosaurus rex and Brachylophosaurus canadensis [1,2], indicating that proteins could be preserved over geological time spans.

Introduction
Homogenization and extraction from small tissue samples (<5.0 mg) requires scaled-down protocols that minimize sample dilution and loss. Here we describe a simple and rapid method for the efficient extraction of protein from small solid tissue samples using pressure cycling technology (PCT) and PCT μPestles.

Extraction of total proteins from tissue has generally been limited by the poor solubility of many proteins in traditional extraction buffers. This has been especially true for lipid-rich samples such as adipose tissue, but also for many other types of samples. Traditional detergent-based sample preparation methods may not adequately dissociate all proteins, especially hydrophobic proteins, which may be tightly associated with membrane lipids. Isolation of these proteins is often very inefficient, because the bulk of membrane proteins are often discarded in the insoluble fraction after extraction. As a result, proteomic analysis of tissues is often biased toward the more soluble proteins. We have previously described a method for efficient extraction of proteins from samples of a variety of mammalian tissues, using pressure cycling technology (PCT) and the novel chemistry of

Pressure BioSciences’ ProteoSolve-SB kit. Here we show that by using the new PCT MicroTubes, the ProteoSolve-SB protocol may be scaled down for use with tissue samples in the 10-20 mg size range. This scaled-down method is compatible with biopsy-size tissue samples.

Introduction
Pancreatic cancer has a very high mortality rate, primarily due to the fact that it is usually diagnosed at an advanced stage (Ranganathan 2009). Early diagnosis of this devastating disease could be crucial for improving treatment options and survival rates. The pancreas, as the site of insulin secretion, is also intimately linked to diabetes. A better understanding of the normal and diseased pancreatic proteome might give researchers better insights into pancreatic function and development in health and dysfunction in various disease states (Tonack et al., 2009; Chen et al., 2007).

Introduction

Protein expression in E. coli is an efficient and commonly used method to generate large quantities of protein for research or therapeutic applications. Unfortunately, proteins expressed at high levels in E. coli are often packaged into inclusion bodies (IBs). These tightly-packed structures have the advantage of being composed of almost pure expressed protein, but the serious disadvantage that the protein is so tightly aggregated that high concentrations of chaotropes or detergents are required to extract soluble protein from the aggregates. These solubilization reagents must then be diluted or removed by buffer exchange, so that the extracted protein can be refolded into its native, functional conformation.

High hydrostatic pressure has shown promise as a means of disaggregating and solubilizing protein aggregates using relatively mild buffer conditions [1-4]. By disaggregating IBs without the high levels of denaturants required under conventional conditions, subsequent protein refolding can be improved.

Here we report that high hydrostatic pressure can be used to efficiently disaggregate proinsulin inclusion bodies in order to extract soluble proinsulin protein. This disaggregation can be carried out in mild buffer conditions at ambient temperature in as little as 5 minutes at 45kpsi.

Introduction

Disaggregation and solubilization of protein aggregates in mild reagents is challenging. Most disaggregation protocols call for protein denaturation in harsh reagents such as detergents, concentrated guanidine-HCl, or 8M urea. Pressure can also be used to denature proteins, and high hydrostatic pressure has shown promise as a means of solubilizing and/or refolding insoluble aggregates due to its effects on electrostatic and hydrophobic interactions – two key components of aggregate formation [2-3]. Disaggregation by pressure works in manner similar to chemical disaggregation, with one significant advantage; pressure-disaggregated proteins do not require extensive clean-up to remove the high concentrations of denaturing chemicals required by conventional methods.Here we report that solubilization of aggregated β-Casein can be enhanced when carried out under high pressure, even in the absence of strong chaotropes. The goal of this work is to provide the user with the best set of starting conditions for pressure-enhanced solubilization of β-Casein or similar aggregated proteins.

Introduction

Protein expression in E. coli is an efficient and commonly used method to generate large quantities of protein forresearch or therapeutic applications. Unfortunately, proteins expressed at high levels in E. coli are oftenpackaged into inclusion bodies (IBs). These tightly packed structures have the advantage of being composed ofalmost pure expressed protein, but the serious disadvantage that the protein is so tightly aggregated that highconcentrations of chaotropes or detergents are required to extract soluble protein from the aggregates. Thesesolubilization reagents must then be diluted or removed by buffer exchange, so that the extracted protein can berefolded into its native, functional conformation.