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PROTEIN SEPARATION While mass spectrometry has become the method of choice for the identification of proteins, several alternative appropraches are being employed for the separation of complex mixtures that precedes their MS analysis. Traditionally, the most fequently applied protein separation strategy has been on two-dimensional polyacrylamide gel electrophoresis, a technique that has the ability to separate up to 10,000 components. While the independent manual digestion of multiple gel spots is time-consuming, robotic digestion can imporve this limitation. In the last few years, enzymatic digestion of useparated protein mixtures followed by spearation of peptides by multidimentional liquid chormatography (LC) has been used as an alterative. Among other beneifts, this LC-based approach lends itself to automated peptide separation together with the acquisition and analysis of MS data. Despite concerns regarding the perils of factionation to proteins, fractionating proteomes for analysis has an overriding advantage in that it simplies them. One's ability detect multiple protein species in a sample depends on one's ability to resolve many peptides from the sample and obtain MS data on as many of them as possible. Selection of a subcellular fraction (e.g., the mitochondria) for analysis reduces the task from analysis of perhaps 25k proteins in a human cell sample to about 1000-2000. There is a better chance of identifying less abundant proteins when they are present in a mixture of 1000 proteins then when they are in a mixture of 14K. Electrophoresis: Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells) to investigate subunit compositions and to purify the protein for subsequent applications. In polyacrylamide gel electrophoresis, proteins migrate due to an electrical field through pores in the gel matrix. Pore size decreases with higher acrylamide concentrations. The migrations of the protein is determined by the gel pore size and protein charges, size and shape. One dimensional gel (1-D) electrophoresis under denaturing conditions (i.e., in the presence of 0.1% SDS) separates proteins based on molecular size. Most proteins bind SDS in a constant weight ratio, leading to identical charge densities for the denatured proteins. Thus the SDS protein complexes migrate in the polyacrylamide gel according to size, not charge. Most proteins are resolved on polyacrylamide gels containing from 5% to 15% acrylamide. The relationship between the relative mobility and log molecular weight is linear over these ranges. After the proteins are solubilized by boiling in the presence of SDS, an aliquot of the protein solution is applied to a gel lane, and the individual proteins are separated electrophoretically. 2-mercaptoethanol is added during solubilization to reduce disulfide bonds. Comparison of reducing and nonreducing gels can provide valuable information about the number of disulfide cross linked subunits in a protein complex. If the subunits are held together by disulfide linkages, the protein will separate in denaturing gels as smaller sized subunits as compared to nonreducing conditions where the protein will separate as a complex. The polyacrylamide gel is cast as a separating gel topped by a stacking gel and secured in an electrophoresis apparatus. After leaving the stacking gel, the protein enters the separating gel which has a smaller pore size, a higher salt concentration and higher pH compared to the stacking gel. The proteins are separated according to either molecular size in a denaturing gel (containing SDS) or molecular shape, size, and charge in a nondenaturing gel. Two-dimensional (2-D) gel electrophoresis separates proteins in the first dimension by isoelectric focusing and in the second dimension by electrophoresis in the presence of SDS. Thus information is obtained not only about size as in one-dimensional gels but also about the charge of the protein. In isoelectric focusing a pH gradient is established using ampholines. Proteins migrate to their isoelectric point (pH) at which there is no net charge. The protein spots can be visualized by staining. An imaging system is used to record an image of the stained gel to provide a record of the protein distribution in the sample. These images can be analyzed, compared, and archived with software packages. A concern with 2D gels is that despite their resolving power, 2D gels do not completely resolve all proteins into single spots. Many spots contain 2-5 proteins. Another concern is that even with the most sensitive stains, there is a limited dynamic range for protein detection. Cellular expression levels of different proteins can differ by as much as a million-fold whereas the dynamic range for protein staining is about 100-2000 fold. Thus, 2D gels typically detect only the most abundantly expressed proteins. In a powerful technique, 2D-SDS-PAGE is combined with a high-throuput MS analytical method (MALDI-TOF MS). In gel digestion is used to cleave the proteins to peptides which are then analyzed by MALDI-TOF MS. The MS data is then analyzed with a peptide mass fingerprinting algorithm and software which identifies the proteins present. A second approach is to use peptide sequence identification by tandem MS. Here, one digests the proteins in the mixture to peptides which are then resolved (at least partially) by chromatography and then electrospray tandem MS. These spectra are mapped to protein sequences from databases with the aid of Sequest or similar search tools. The 2 distingushing characteristics of this approach are 1) the analysis primarily involves working with peptides rather than with proteins and the protein identification is based on the MS-MS fragmentation spectra, rather than on peptide mass fingerprinting. Western blotting is used to identify a specific protein in a complex mixture of proteins. The technique exploits both the efficiency of SDS-PAGE to separate a mixture of proteins into distinct protein bands, adn the ability of immunochemical reagents to interact specifically with a given protein antigen. A typical Western-blotting protocol incluces the following steps: (1) The mixture is first electrophoretically separated on a polyacrylamide slab gel in the presence of SDS. (2) The protein bands are then transferred to a nitrocellulose membrane by electrophoresis. (3) The membrane contianing the protein bands is serially incubated with (a) a suitable blocking reagent to prevent non-specific protein binding, (b) a wash solution to rinse any unbound blocking reagent (c) a probing antibody (anti-protein-of interest antibody) that forms a specific immune complex with Protein-of interst, (d) additional wash solution to remove any unbound antibody, (e) an enzyme-linked antibody that binds specifically to the Fc region of the anti-protein-of interest antibody, (f) additional wash solution to remoe any unbound enzyme-lined anntibody and finally, (g) a substrate solution, which int he presence of the enzyme, yields an insoluble, colored product that precipitates at the site of the immune complex, thereby rending the Protein of interest band visible. Chromatography Column Chromatography can be used to fractionate proteins. The mixture of proteins in solution is passed through a column containing a porous solid matrix. The 3 types of matrices used are 1) Ion-exchange chromatography, 2) gel-filtration chromatography and 3) affinity chromatography 1) Ion-exchange chromatography uses an insoluble matrix that carries ionic charges that retard the movement of molecules of opposite charge. There are 2 types. anion and cation. Protein purification using ion-exchange chromatography usually employs positively charged anion exchangers because the majority of proteins are negative charged at neutral pH (i.e., have a low isoelectric point). 2) Gel-Filtration Chromatography uses a porous matrix. Molecules that are small enough to penetrate into the matrix are delayed and travel more slowly through the column. 3) Affinity Chromatography can be used to isolate and identify proteins that interact physically. The method takes advantage of a protein's binding site and its ligand. The ligand, when immobilized, attracts the protein from a mixture, while other molecules are washed away. Most proteins have specific ligands; For example, enzymes have subtrates and cofactors. Beads coated with Avidin: One often used method of affinity chromatography is to use a column containing avidin-coated beads. A sample is incubated with an antibody that is conjugated to biotin. When the sample is passed through the column the biotin binds to the avidin. This has been used to isolate hematopoietic stem cells by conjugating CD34 to biotin and passing them through the column. High Pressure Liquid Chromatography (HPLC):
Multidimensional Liquid Chromatography: A sample is fed through a HPLC using a particular type of separation method, and peaks are selected and fed into a second column using a different mode of separation. To capture interacting proteins, a target protein is attached to polymer beads that are packed into a column. Cellular proteins are washed through the column and those proteins that interact with the target adhere to the affinity matrix. These proteins can then be eluted and their identity determined by mass spectrometry or another method. |
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