Proteomics/Protein Sample Preparation/Protein Handling and Storage

Protein Handling and Storage
Over time, changes will take place in protein structure that could potentially alter experimental results. There are also many problems that can arise from improper handling of proteins. Contaminants in samples can cause results to be skewed, and may even damage equipment. However, there are many steps that can be taken in the protein handling and storage processes that help to minimize any damage and in turn, maximize accuracy of results.

Sterile Work Environment
Whenever handling protein samples, it is very important to wear gloves. Often times gloves are used to protect us when handling harmful substances, but the gloves are also used to shelter the sample from any foreign proteins or chemicals found on our hands. Contamination could not only lead to unexpected results but could potentially damage our sample. For this same reason it is also very important to always use sterile containers, pipette tips, etc.

Flash Freezing
At some point it may be desirable to store a protein for use months down the road; most often, this is achieved by freezing. By storing proteins at temperatures as low as -20 degrees celcius, the structural components of the molecule can be preserved until its use, assuring functionality. However, if the freezing process is not carried out correctly, the protein can actually be subjected to drastic changes in pH as well as in salt concentration. To avoid these environmental changes over time, the sample can be "flash frozen" by dipping the tube containing protein sample in a mixture of dry ice, ethanol and acetone. After being frozen, the sample can then be rapidly thawed in a beaker of water.

Protein Loss
While performing any proteomics experiment care must be aware of potential for loss of proteins or peptides due to chemical properties of those proteins in a sample. For example, it is not uncommon for hydrophobic proteins to stick to the walls of test tubes or chromatography columns in solution. If a protein of interest is known to be hydrophobic, the glass ware to be used can be silanized or salanized glassware can be purchased to prevent loss of the protein.

Working with Small Samples
Micro-biopsies, laser capture micro-dissection, and stem cell therapy only provide small, non-conventional sample sizes, and in order to take advantage of these small samples, a less wasteful sample preparation is needed. Trifluoroethanol provides a viable alternative to accomplish this.

Hydrostatic Pressure Cycling Technology
Pressure cycling technology (PCT) alternates pressure in a reaction vessel. For example, a sample is placed in an air-tight vessel and next the pressure in the vessel is alternated between very high pressure and room pressure. When pressure drops from very high down to room levels, compressible molecules (such as lipids) tend to dissociate. One example of useful dissociation is that of bilayer lipid membranes, which results in the release of proteins and other metabolites from the bilipid layer. PCT-assisted liquid-liquid extraction leverages PCT to fraction a sample into different classes, using class-specific immiscible solvents within the same reaction vessel. The initial solvents are combined with the raw sample, then the pressure is raised. As the pressure peaks, the solvents mix, forming a temporary solvent, and the sample dissolves in this new temporary solvent. The pressure is lowered, and the temporary solvent separates back into the initial solvents, fractioning the dissolved sample in the process (the different classes of sample molecules being more soluble in a particular initial solvent). At room pressure, each class of sample molecules are found isolated within each of the initial, immiscible solvents.

Development and Evaluation of a Micro- and Nano-Scale Proteomic Sample Preparation Method
Haixing Wang,Wei-Jun Qian,Heather M. Mottaz,Therese R.W. Clauss,David J.Anderson,Ronald J.Moore,David G.Camp,Maria Pallavicini,Desmond J.Smith and Richard D.Smith J Proteome Res. 4(6):2397–2403 (2005)

Main Focus

Using an organic co-solvent (TFE) instead of traditional detergents minimizes sample loss.



Summary

Micro-biopsies, laser capture micro-dissection, and stem cell therapy only provide small, non-conventional sample sizes, and in order to take advantage of these small samples, a less wasteful sample preparation is needed.

Typical protocols for proteomic sample processing usually involve a strong denaturant such as urea or guanidine hydrochloride combined with different types of detergents. These methods are rapid, however, the detergents or salts need to be entirely removed from the sample before the LC-MS analysis. If they are not properly removed, the level of detection sensitivity may not be enough for the peptides. These procedures show their weaknesses when working with sample sizes less than a microgram, because some of the sample is inevitably left behind when removing the detergents.

To get around the issues involved with using detergents, a different sample preparation technique using only a single tube was used. The technique used trifluoroethanol (TFE), in a hypotonic aqueous buffer as a replacement for the detergents. TFE is better because it improves protein solubility and denaturation, and it readily evaporates, so there’s no need to remove the TFE when no longer needed. So using TFE in the place of detergents removes the cleanup step, eliminating the waste involved with removing the detergent.

An experiment was conducted to test this hypothesis using two different mouse brain tissue samples. One was disrupted in a buffer of denaturants and CHAPS, and the other one in a hypotonic buffer containing 50% TFE. The chromatogram figures of the two samples indicated that the presence of detergent in the first sample has suppressed the signals of peptides that are co-eluted with the detergent. A larger number of proteins were identified using the TFE protocol rather than CHAPS protocol. Also, TFE proved to be more efficient than the traditional detergent based protocol even with processed MCF cells, with a larger number of proteins identified than when using traditional methods.

The TFE based protocol proved to be more efficient in micro and nano scale sample processing than traditional methods. The main advantage in using the TFE based protocol is that extra cleanup steps are eliminated and the whole process is carried out in a single tube, hence there is no manhandling and no loss of sample. Other advantages of TFE are that it improves protein solubilization, increases protein denaturation, and finally it can be removed by tryptic digestion without performing any extra clean up step. The TFE based protocol also provides better sample recovery and better reproducibility.

New Terms


 * Organic Solvent: A chemical compound (usually liquid) containing carbon to dissolve another substance (the solute). ( http://www.knowledgebank.irri.org/glossary/Glossary/O.htm )
 * Trifluoroethanol: The organic compound with the formula CF3CH2OH. Also known as TFE or trifluoroethyl alcohol, this colourless, water-miscible liquid has a smell reminiscent of ethanol. ( http://en.wikipedia.org/wiki/Trifluoroethanol )
 * Liquid Chromatography: A separation method based on the distribution of sample compounds between a stationary phase and a liquid mobile phase. ( http://www.nhml.com/resources_NHML_Definitions.cfm )
 * Solid Phase Extraction: A separation process that is used to remove certain compounds from a mixture of impurities based on their physical and chemical properties. ( http://en.wikipedia.org/wiki/Solid_phase_extraction )
 * Zwitterion: A chemical compound that carries a total net charge of 0, thus electrically neutral but carries formal positive and negative charges on different atoms. ( http://en.wikipedia.org/wiki/Zwitterionic )
 * Voxellation: High-throughput acquisition of multiple volumetric images of brain gene expression. ( http://labs.pharmacology.ucla.edu/smithlab/publications_files/pdf/Singh_JNM_2003.pdf )

Course Relevance

Sample preparation efficiency can be a large problem when trying to detect different proteins in a proteome, because if a proteome only has a tiny amount of a certain protein to begin with, then any loss of the sample during preparation may mean the loss of some or all of that protein. Therefore, learning a variety of different sample preparation methods, preferably the most efficient methods, is useful to students hoping to work with and prepare proteomic samples.

Tissue Fractionation by Hydrostatic Pressure Cycling Technology: The Unified Sample Preparation Technique for Systems Biology Studies
Gross V, Carlson G, Kwan AT, Smejkal G, Freeman E, Ivanov AR, Lazarev A. Journal of Biomolecular Techniques 19:189-199 (2008)

Main Focus

The authors present a novel, detergent-free, concurrent sampling process for high yield extraction of multiple classes of biological molecules from the same sample, using pressure cycling technology (PCT) as the basis of the new process. A significant advantage of the new process is that the samples may either be used directly in another (compatible) process for further analysis (such as MS), or require very minimal cleanup before additional analysis. Relevance to systems biology studies is emphasized.



Summary

Given the widening popularity of systems biology studies, it's become increasingly important to improve the consistency and quality of the samples under analysis. Many current high quality sampling techniques are geared toward a single type of analysis (DNA/RNA, or protein, or lipids). Concurrent sampling techniques are important, in particular with regard to systems biology studies, because of the way that information from several classes of molecules must be correlated during analysis. Concurrent techniques are also critical where small sample size is a constraining factor (for example, human biopsy tissue).

The authors have developed a new concurrent sampling protocol that cleanly separates DNA, RNA, proteins, and lipids using detergent-free processes. The new protocol leverages pressure cycling technology (PCT), as well as a proprietary set of partitioning reagents that separate molecules by class. PCT destabilizes molecular interactions by quickly alternating pressure, from very high to low. High pressure mostly affects compressible molecules (lipids), which subsequently dissociate when the pressure is lowered. PCT does not impact covalent bonds, so DNA, RNA, and proteins remain intact while lipid bilayers are dismantled.

PCT-assisted liquid-liquid extraction is another PCT-based technique that's used to partition a sample given a set of partitioning solvents. For example, two solvents are combined with the sample, the solvents being immiscible. Pressure is then increased, and the solvents begin to merge. When the pressure is high enough, the solvents mix and form a temporary tertiary solvent in which the sample dissolves. The pressure is then lowered, and the tertiary solvent separates back into the initial two solvents, particles of the original sample being partitioned into either of the two solvents. When the pressure is back to “normal”, the original sample has been cleanly partitioned into the separate solvent phases. In previous works, the authors have evolved the above PCT-based techniques, with some commercial success. The newly developed ProteoSolve-SB protocol builds upon this previous work such that the new protocol cleanly separates DNA, RNA, proteins, and lipids from a single sample.

ProteoSolve-SB is compared to current protocols, including Thizol, AllPrep, and PARIS. Thizol shows good DNA recovery, but suffers from slow extraction and cleanup from the organic phase, as well as from possible protein modification. Thizol works well with relatively low sample concentrations (less than 10% of volume), whereas ProteoSolve-SB is compatible with sample concentrations up to 25% to 30% of the overall volume. Sample concentration is an important factor since subsequent precipitation procedures are more efficient with higher concentrations of the sample.

AllPrep had RNA recovery volume similar to Thizol and ProteoSolve-SB, however it's a more expensive process and required multiple columns for the test procedure. AllPrep also suffers from the side-effect of potential protein loss: many proteins may not unbind from RNA, so they may not be recoverable. Also, the proteins that are recovered must be cleaned before SDS-PAGE; the cleaning process may incur additional protein loss.

PARIS recovered less than half the RNA as compared to the other tested protocols, which was expected since only half the sample was used for the RNA analysis: the other half was used for protein analysis. In addition to low yields of RNA, the recovered proteins required additional cleanup (dialysis, filtration).

ProteoSolve-SB has been shown to yield high recovery of DNA, RNA as well as a significant recovery of protein and an simplified recovery of lipids (fewer steps than other methods, less hands-on time). Sample homogenization via PCT yields more consistent samples, as opposed to sonication or grinding in liquid nitrogen. Once centrifuged, minimal cleanup is required for analysis: the proteins are ready to go after drying; lipids are ready to go for MALDI-TOF MS, and; DNA, RNA may be extracted from the solid phase using common, compatible extraction protocols. Future work will focus on scaling down the process further for applications such as needle biopsies, as well as for cell cultures derived from individual stem cells.

New Terms


 * Biopsy: The removal and examination of a sample of tissue from a living body for diagnostic purposes.
 * Dialysis: Separation of substances in solution by means of their unequal diffusion through semipermeable membranes.
 * Hydrostatic: Of or relating to fluids at rest or to the pressures they exert or transmit.
 * Sonication: Disruption of cells or DNA molecules by high frequency sound waves. a.k.a. ultrasonication.
 * Systems biology: A field that seeks to study the relationships and interactions between various parts of a biological system (metabolic pathways, organelles, cells, and organisms) and to integrate this information to understand how biological systems function.

Course Relevance

This new process provides a low-loss rate for proteins in the original sample, as well as avoids detergents and other solvents that may change the protein before it can be analyzed further downstream. It's relevant to proteomics because it provides another method of extracting proteins from cell tissue in a manner that reduces the loss and/or the contamination of the proteins from the original sample. It's also a relevant process for samples that are unique or hard to obtain.

Cleanup of detergents
Cleanup of protein detergents: A gas chromatographic method for quantification of detergents frequently used in membrane protein structural studies

Gas Chromatography http://www.separationsnow.com/coi/cda/detail.cda?chId=3&id=20260&type=Feature&page=1/ (2009-01-26)

Main Focus The traditional method of protein extraction involves use of detergents. Though they affect the stability of the proteins, they were used in the extraction for quite a long time until the advantages of organic solvents are known.

Summary

Structure of proteins are studied outside the functional environment in conditions different to normal conditions. The most conventional method is the use of detergents in the extraction, purification, concentration and crystallization of proteins. But the concentration of detergents have a strong impact on the stability of the proteins. The procedure of measuring the amount of detergent associated with the protein was illustrated by using four detergents decyl maltoside, dodecyl maltoside, dodecyl phosphocholine and lauryldimethylamine N-oxide. Separation of the detergents was carried out at a temperature gradient of 200-280C and low injection volumes of 1 microlitre. The retention times for all these four detergents were suitable for the separation and identification of detergent mixtures. Separations were carried out under the experimental conditions and the resultant calibration curves matched with the quadratic regression curves. Thus this is one of the most easy methods for the separation of detergent mixtures from the proteins.



New Terms


 * Amphipathic: Molecule containing both polar and non polar portions. (http://www.biology-online.org/dictionary/Amphipathic)
 * Quadratic regression: It is a process by which equation of a parabola of best fit is found for a set of data. (http://calculator.maconstate.edu/quad_regression/index.html)
 * Centricon filtering: Centricon filtering devices provide fast efficient concentration and desalting of macromolecules by ultrafiltration.(http://www.millipore.com/userguides/tech1/p99259)
 * Elution buffer: Elution buffer is used to was out any left over proteins from the previous experiment.(http://wiki.answers.com/Q/What_is_an_elution_buffer)

Course relevance
 * Gas Chromatography: A process by which the components of a mixture are separated from one another by volatilizing the sample into a carrier gas stream and passing the gas through a column containing a substance that selectively retains (absorbs) and releases the volatile constituents. (http://www.biotechmedia.com/definitions-g.html)

Detergent based extraction is used for the extraction of proteins.The advanced method in this is extraction of proteins using organic solvents for which separation is not needed usually they are evaporated.

Next Generation Pharmaceutical
Pressure Cycling Technology (PCT): a Novel Sample Preparation Approach to Biomarker Discovery and Drug Development in Lipid-Rich Samples

Pressure Biosciences http://www.ngpharma.com/article/Issue-9/Drug-Discovery/Pressure-Cycling-Technology-PCT-a-Novel-Sample---Preparation-Approach-to-Biomarker-Discovery-and-Drug-Development-in-Lipid-Rich-Samples/ (2009-03-24)

Main Focus

PCT will improve the rate of discovery for proteomics-based research by providing practitioners with a technology that safely and efficiently fractions biological samples into specific biological classes (proteins, lipids) that may, in some cases (especially for proteins), be fed directly into common downstream analysis processes.

Summary

Major diseases in the US continue to be targets of biological research: namely obesity, heart disease and diabetes. The relationships between such diseases are complex as are the analyses of the biological mechanisms. Improved processes and technology are needed to battle these diseases, and of particular importance is more efficient drug discovery and drug development cycles. Such improvements will increasingly support complex research efforts, including the use biomarkers and proteomics to drive discovery forward.

There are some important technological challenges to address in the proteomics space, including the need for processes which are increasingly sensitive to lower protein concentrations as well as in tissue proteomics, where proteins of interest are found primarily in lipids. Sample preparation techniques play a large role in the outcome of proteomics experiments, and many current preparation techniques have problems such as safety, contamination, long processing times and are not easily automated. Compounding the problem is that some techniques are only useful for specific sample types. As a consequence, experimental results are not easily, reliably reproduced.

Proteins found in high lipid concentration tissue are critical to our understanding of important diseases. The use of detergents presents a challenge because not all the protein in a sample may be exposed, especially if the lipid concentration exceeds sixty percent and the sample is prepared in an aqueous solution. Furthermore, detergents are not always compatible with subsequence analysis procedures. There have also been cases where proteins of interest have been discarded with pellets as cell debris. These types of challenges can have negative impacts on the pace of research.



Pressure cycling technology (PCT) addresses the aforementioned concerns. In a single step, using all organic (not detergent-based) solvents, lipids and proteins are dissociated and partitioned in a manner that is very safe for the researcher. PCT-based liquid-liquid extraction is leveraged to capture dissociated molecules in different solvent fractions. Samples are added to a combination of immiscible solvents. Next, PCT makes use of high pressure in the reaction vessel to break apart and mix samples with liquid solvents. Upon the release of the high pressure, sample molecules are fractioned in the initial liquid solvents. The reaction vessel in PCT (special reaction tubes) are designed to withstand high pressure, isolate the sample and solvents from contaminants as well as to provide safety to the researcher. PCT is especially good at releasing proteins from their captivity in lipid tissues.

In a study between Pressure BioSciences and Harvard School of Public Health, the results of proteomic analysis of adipose tissue were deemed useful for the mechanistic studies of diabetes and obesity. In the results of the study, PCT is reported to have higher protein yields compared to more “traditional” techniques. PCT results were high yield and efficient to come by. PCT was also compatible with the downstream protein identification method (HPLC-ES/MS). PCT processes are also highly reproducible.

Other applications may include those dealing with neurological conditions, such as Alzheimer's, Parkinson's, Multiple Sclerosis, hyperactivity, depression and/or schizophrenia. Neurological disorders make an interesting research target because myelin, which coats a major portion of neuron cells, has a protein/lipid ratio of about three to seven. Phospholipids, which tend to be membrane proteins, are important for neuron regulation – as such, phospholipids also make an interesting research target.

New Terms


 * Adipocyte: Animal connective tissue cell specialized for the synthesis and storage of fat. Such cells are bloated with globules of triglycerides.
 * Biomarker: A biochemical feature or facet that can be used to measure the progress of disease or the effects of treatment.
 * Cavitation: The formation and instantaneous collapse of innumerable tiny voids or cavities within a liquid subjected to rapid and intense pressure changes. Cavitation produced by ultrasonic radiation is sometimes used to effect violent localized agitation. Cavitation caused by severe turbulent flow often leads to cavitation damage.
 * Delipidation: The removal of lipids or lipid groups, often from a protein.
 * Immiscible: Incapable of being mixed without separation phases. Water and petroleum oil are immiscible under most conditions, although they can be made miscible with the addition of an emulsifier.

Course Relevance

PCT is relevant to proteomics because it is the basis of an efficient, safe, automated, reproducible sample preparation technique, of particular importance with respect to protein extraction from lipid tissues.