Proteomics Application Notes

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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.

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
Proteomic profiling of mitochondria has the potential to provide insights into mitochondrial functions associated with aging, various metabolic states and diseases such as cancer, diabetes and cardiovascular disease [1]. Rapid and reproducible isolation of intact mitochondria is crucial for efficient enrichment and subsequent proteomic analysis of low abundance mitochondrial proteins [2]. Here we describe a system for the isolation of intact mitochondria from rat PC12 cells using pressure cycling technology (PCT).