Peptides and proteins can be separated and purified based on their size or charge, and then structurally analyzed using several methods.
Gel electrophoresis is a technique used to separate molecules based on size and charge, and can be used to separate proteins as whole molecules or fragments. The proteins/peptides are loaded into a slot near the negative electrode of a the porous gel matrix. Proteins are most commonly run in polyacrylamide gel electrophoresis (PAGE) gels. Because proteins are generally negatively charged, they are pulled toward the positive electrode at the opposite end of the gel. Smaller molecules move through the pores in the gel faster than larger molecules, and this difference in the rate of migration separates the fragments based on size. There are molecular weight standard samples that can be run alongside the molecules to provide a size comparison.
Proteins in their natural, tertiary-structured state can be run on native-PAGE gels – this separates proteins by both size and conformation. Larger, bulkier proteins will run toward the positive cathode more slowly. Proteins can also be chemically denatured before running them on the gel – in this case, proteins are separated exclusively by size. The chemical SDS denatures proteins, and so denaturing SDS-PAGE gels are used for these experiments. Proteins can be further denatured by the addition of a reducing agent that breaks down the disulfide bonds between cysteine residues (these are not fully broken by SDS alone).
Samples of proteins and peptides can also be separated by size-exclusion chromatography, in which molecules in solution are separated by their molecular weight. This process is usually performed within a column, which typically consists of a hollow tube tightly packed with polymer beads containing pores of different sizes. As the solution travels down the column some smaller particles get stuck in the pores. The larger molecules simply pass by the pores because they are too large to enter the pores. Larger molecules, therefore, flow through the column more quickly than smaller molecules, so smaller molecules have longer retention times.
Ion exchange chromatography is a technique used to separate molecules (or proteins) according to their charge. This method is based on the attraction between oppositely charged ions, so a cationic stationary phase is used to separate anions in cation-exchange chromatography, and an anionic stationary phase is used to separate cations in anion exchange chromatography. The molecules of interest that were bound to the oppositely-charged stationary phase can then be drawn out (eluted) and collected by running higher concentration of ions through the column or changing pH of the column.
Affinity purification of protein complexes involves a specific protein (the bait) being manipulated to express an affinity tag. The tag serves as a tool to purify the bait protein and associated proteins by affinity chromatography. This occurs when the affinity tag on the protein is attracted to a highly specific antigen/nucleic acid in the chromatography process.
Once these samples have been separated they can be analyzed by various quantitative methods:
Mass spectrometry is used to identify and determine the composition of a molecule. An energy source causes the molecule to break into smaller fragments which are detected by their mass to charge ratio.
X-ray crystallography enables scientists to determine the three-dimensional structure of a protein crystal at atomic resolution by analyzing the patterns created by deflected X-rays.
Nuclear magnetic resonance (NMR), uses the magnetic properties of atoms to determine the composition and three-dimensional structure of proteins in solution. NMR analysis is much more complex for proteins than for smaller organic molecules.
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• Gel electrophoresis and chromatography methods can be used to separate mixtures of proteins and peptides for analysis.
• Gel electrophoresis can be done using native-PAGE or SDS-PAGE (with or without a reducing agent), depending on whether you are interested in the native conformation or the denatured state of the protein.
• Size-exclusion chromatography separates molecules by their molecular weight. Larger molecules bypass pores in the stationary phase and elute first.
• Ion exchange chromatography is a technique used to separate molecules (or proteins) according to their charge. An oppositely-charged stationary phase holds on to the charged molecule of interest, which is later collected by changing the pH of the column.
• Affinity purification separates a specific protein based on an affinity tag designed for that chromatographic process.
• Quantitative structural analysis of proteins can be done using mass spectrometry, NMR, or X-ray diffraction.
gel electrophoresis: a technique used to separate molecules based on size using electric charge
native-PAGE: a technique for separating proteins based on size and conformation
SDS-PAGE: a technique for separating proteins based on size only, because proteins are denatured
reducing: refers to the chemical reduction of the disulfide bonds between cysteine residues in a protein
elution: the process of drawing out fractions from a column
affinity tags: molecules appended to proteins so that they can be purified from their crude biological source using an affinity technique
mass spectrometry: an analytical technique that measures the mass-to-charge ratio of ions and molecular fragments
X-ray diffraction: an analytical technique for determining the atomic and molecular structure of a crystal using X-rays
NMR: a spectroscopic technique to observe local magnetic fields around atomic nuclei to determine molecular structure
agarose: a polysaccharide linear polymer made up of the repeating unit of agarobiose used to make a gel matrix
molecular weight: the molecular mass of a substance
size-exclusion chromatography: a chromatographic method in which molecules in solution are separated by their size