Proteomics/Protein Sample Preparation/Sample Preparation for Electrophoresis

Sample Preparation for Electrophoresis
Polyacrylamide Gel Electrophoresis (PAGE), described in detail in Chapter 4.2, is a process by which molecules, typically proteins, are separated by their mass. By assuming the speed at which a protein travels through a gel matrix is directly related to its mass, that is, the total matter in the protein, it is crucial to ensure the shape of the protein is not affecting its speed. As such, a number of sample preparation techniques have been refined to confer similar shapes on all proteins in a sample. Furthermore, protocols have been refined to maximize the consistency of mass related characteristics. This section aims to confer the purpose and procedures of a number of these techniques involved in preparing a sample for gel electrophoresis.

SDS Treatment:
Sodium dodecyl sulfate (SDS) is an anionic detergent used to denature proteins prior to gel electrophoresis. As stated above, it is necessary to denature all proteins in a sample in order to separate them solely on their mass in a gel matrix. SDS efficiently breaks down secondary structures like alpha-helices and beta-sheets (both primarily comprised of hydrogen bonds) as well as many tertiary structures. However, SDS does not break down any of the disulfide bonds that participate in many tertiary structures; treatment with DTT, described below, is often necessary to break down disulfide bonds. SDS is found to be particularly useful in 2-dimensional electrophoresis (2DE). As mentioned in Chapter 4.2, 2DE involves an initial stage of Isoelectric Focusing (IEF) which results in all proteins finding their pI on the IEF strip resulting in all proteins having a net charge of zero. Due to the nature of PAGE, a negative charge is necessary to induce movement of the proteins through the gel matrix. Fortuitously, treatment of a protein sample with SDS not only denatures all proteins in the sample, it also binds all proteins and confers its negative charge to the bound proteins. It is seen that SDS binds a protein in a manner proportional to the mass of that protein which results in a negative charge directly related to the protein’s mass; further supporting a separation based on mass alone.

DTT Treatment:
Dithiothreitol (DTT) is a reducing agent typically used to break down the disulfide bonds contributing to tertiary structure which SDS was unable to affect, further denaturing the protein to deemphasize the role of protein shape in PAGE. DTT reduces a disulfide bond by two sequential thiol-disulfide exchange reactions resulting in DTT becoming a six-member ring structure. 2-mercaptoethanol (ME), another disulfide reducing agent, is commonly used in lieu of DTT. Furthermore, it should be noted that while both DTT and ME sufficiently reduce disulfide bridges in proteins, there is a propensity for them to reform. Thus the protein sample is commonly treated with 2-Iodoacetamide, an alkylating sulfhydryl reagent, which binds covalently to free sulfides and prevents disulfide bridges from reforming.




 * Dithiothreitol (DTT):


 * 2-mercaptoethanol (ME):

Protein Extraction from Yeasts
To extract proteins from yeast, the collected yeast cells are resuspended in a buffer containing NaOH, 2-mercaptoethanol, and SDS and immediately heated to 90oC. Then the sample is neutralized using a Tris-HCl buffer. Glycerol and bromophenol blue is then added so that the final sample is contained in a standard SDS-PAGE buffer. The combination of both the heat and the buffer in effect destroys all cell walls and at the same time makes soluble the proteins needed to be extracted.

Varying the concentration of SDS, boiling time, and the addition of solubilizing reagents can change and optimize the extraction of yeast proteins. The addition of deoxycholate or urea to the above protocol was found to optimize the extraction of certain proteins but decreased the optimization of others. The above parameters are optimal for the extraction of most of the proteins expressed in S. cerevisiae, but not for all proteins. To this date, there is no one universal buffer or protocol that can yield all expressed protein from the yeast cell.

Optimized Protein Extraction for Quantitative Proteomics of Yeasts
von der Haar T PLoS One 2(10):1-8 (2007)

Main Focus

The main focus of this article is to find the optimal extraction technique for yeast proteins. The von der Haar group has found an optimal technique for most of the yeast proteins but not all expressed proteins.

Summary



The abundance data that come out of published papers for any protein can vary from group to group. This is due to the varying efficiencies of the extraction procedures that they use. The von der Haar group has found a protein extraction procedure that can extract most of the proteins in the yeast S. cerevisiae. The original protocol that they found to be the best calls for the use of 0.1N NaOH as the resuspension buffer, incubate for several minutes, then boiled in standard SDS-PAGE sample buffer. The problem with this procedure is that when yeast is suspended and incubated in 0.1N NaOH not all the cell walls are destroyed. Many yeasts cells are still intact. This can lead to the yeast cells to express proteins not normally expressed solely under the environment of study but due to the NaOH environment. The von der Haar group has devised a new protocol that fixes this problem.

The new protocol calls for the extracted yeast cells to be resuspended in a buffer containing NaOH, 2-mercaptoethanol, and SDS and immediately heated to 90oC. Then the sample is neutralized using a Tris-HCl buffer and glycerol and bromophenol blue is then added so that the final sample is contained in a standard SDS-PAGE buffer. The combination of both the heat and the new buffer in effect destroys all cell walls and at the same time makes soluble the proteins needed to be extracted.

The von der Haar group has also found that varying the concentration of SDS, boiling time, and the addition of solubilizing reagents can change the optimization of protein extraction. The optimal concentration of SDS that they have found was 2%. The optimal time of boiling is 10 minutes at 90oC. These parameters are optimal for the extraction of most of the proteins expressed in S. cerevisiae, but not for all proteins. The addition of 1% deoxycholate increased the efficiency of the extraction of Sup45p and Rpl25p, but decreased the efficiency of Sup35p. The addition of 8M Urea increased the efficiency of extraction of Sup35p but decreased the efficiency of all the other proteins.

The von der Haal group did find the optimal protocol to extract most of the proteins expressed in S. cerevisiae, but not for all proteins. The addition of other solubilizing agents can be optimal for certain proteins but can significantly decrease the solubilization of others. It is clear that there can be no one universal buffer or protocol that can yield all expressed protein for the yeast cell.

For more descriptive steps, refer to Figure 1 of the article.

New Terms


 * proteome: The set of proteins expressed by the genetic material of an organism under a given set of environmental conditions ( http://www.answers.com/topic/proteome-2 )
 * cell density: The number of cells per unit volume ( http://www.oilgae.com/ref/glos/cell_density.html )
 * YPD (Yeast Peptone Dextrose): The media that contains peptone, dextrose and other material used to grow S. cerevisiae (http://www.clontech.com/products/detail.asp?tabno=2&catalog_id=630409&page=all)
 * Stationary Phase: Also known as the G0 phase, is a response to the lack of nutrients or starvation. The yeast cell can prolong its life by altering its structure and it metabolism. (http://www.rose.brandeis.edu/PRLab/projects/yeast/yeast.html)
 * alkaline lysis: a protocol using NaOH to break open cells in order to isolate DNA or proteins (http://bitesizebio.com/2007/11/07/the-basics-how-alkaline-lysis-works/)

Course Relevance


 * In the proteomics class we have learned how the analysis of protein expression under certain conditions could be quantified by experiments such as Mass Spectrometry or gel electrophoresis. But in order to correctly analyze the proteins expressed there must be a technique which will extract all if not most of the proteins.  This paper describes the optimal way to extract most proteins specifically for yeast.

Yeast Cell Lysates
The Gottschling Laboratory at Fred Hutchinson Cancer Research Center

http://labs.fhcrc.org/gottschling/Yeast%20Protocols/pprep.html (03/29/09)

Main Focus

The main focus of the website is to provide protocols and other information about the Gottschling Lab, a laboratory focused on cancer research.

Summary

The Gottschling laboratory is interested in studying yeast, specifically S. cerevisiae, because of its link to cancer. When yeast cells enter the middle to late stage of their lifespan, they experience genomic instability. This genomic instability can also be seen in humans due to increased age and cancer. The Gottschling group believe that there is a link between the transition experienced by yeast and the increased age and occurrence of cancer in humans.

The website provides many different protocols along with a protocol for extracting yeast proteins. They list three different ways to prepare yeast proteins for direct gel electrophoresis. The first uses a SUMEB buffer along with a protease inhibitor in order to extract yeast protein. The website provides the ingredients needed to make the SUMEB buffer. The second protocol uses only sample buffer and the website also provides the recipe for the sample buffer. The third protocol that they have listed is the use of glass beads along with sample buffer.

Refer to the website link for a more detailed description of the steps for rapid protein preparation.

Course Relevance


 * In the proteomics class we have learned how the analysis of protein expression under certain conditions could be quantified by experiments such as Mass Spectrometry or gel electrophoresis. But in order to correctly analyze the proteins expressed there must be a technique which will extract all if not most of the proteins. This website gives several different protocols in order to extract yeast proteins as a sample for gel electrophoresis.