The primary attributes used to identify proteins include the protein's mass and apparent mass, its isoelectric point, and its N-and C-terminal sequence tags. A protein's mass and its apparent mass are probably the most common characteristics used. Protein mass is determined by adding the total mass of all the amino acids in the protein to the mass of any molecules added through post-translational modification. A protein's isoelectric point is the pH at which it is neutrally charged. A protein's N-and C-terminal sequence tags are short sequences of amino acids on either end of the protein. Since there are twenty different possible amino acids at each position in a protein, a peptide of only four or five amino acids in length is likely to be unique to a specific protein. There are 160,000 (204) combinations of sequences that are four amino acids long.
The most commonly used laboratory techniques in proteomics are two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spec-trometry. These techniques have been modified for use in proteomics. Both can be used in combination with more traditional protein separation techniques, including column chromatography.
Starting in the late 1990s, several companies also started developing "protein chips," another strategy for studying proteomes and other complex protein mixtures. These chips allow a researcher to collect minute quantities of proteins that bind to specific molecules on their surface. By 2001, some companies announced they were developing "antibody chips" onto which antibodies will be attached. The antibodies can then be used as probes to capture and quantify specific proteins found in complex mixtures.
The use of 2-D PAGE allows the simultaneous separation of thousands of proteins, and the technique is still a key tool in proteomics technologies. The first dimension of protein separation on the gel is by isoelectric focusing, in which proteins are separated along a pH gradient until they reach a stationary position, where their net charge is zero.
The second dimension of separation on the gel is by molecular mass. Sodium dodecyl sulphate (SDS) is applied, and it binds to all the proteins. This provides the proteins with a uniform charge along their length, so that they will migrate across the gel according to their molecular mass when a current is applied. After the 2-D PAGE is run, the gel is stained. The result is a two-dimensional map consisting of hundreds or thousands of protein spots.
Since the early use of 2-D PAGE in the early 1970s, a number of modifications have been made to make gels more reproducible and more amenable to the higher-throughput use necessary for proteomics applications. However, 2-D PAGE is still something of an art form, and high-quality, reproducible results are difficult to obtain except in the hands of very experienced users. The technology needs to be further simplified to allow casual and novice users to obtain reproducible, quality results.
Mass spectrometry is an analytical technique that very accurately measures the mass of proteins and peptides. There are two common types of mass spectrometry. The first type, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, can be used to analyze proteins that are embedded in solid samples and measures their mass in a flight tube. The second type, electrospray ionization mass spectrometry, can be used to analyze proteins that are in a liquid solution and measures their mass in either a flight tube or in a device known as a quadrupole. There are also other variations on these techniques.
Mass spectrometry is commonly used for peptide mass fingerprinting. In this process, a protein sample is isolated by 2-D PAGE and cut with an enzyme that specifically targets particular amino acids. Mass spectrometry is used to measure the masses of the resulting cut pieces, or peptides. These masses can be thought of as a fingerprint that can be compared to the fingerprints of proteins whose amino acid sequences have already been analyzed and stored in a database.
To determine the fingerprints of proteins that have already been sequenced, a computer program determines the amino acid composition, and thus the masses, of the pieces that would result if those proteins were also cut by the same enzyme. A list of proteins is generated from the database, sorted by how many peptides they share with the unknown experimental protein.
There are also technologies, including the yeast two-hybrid system, that can be used to study interactions between proteins. These approaches complement 2-D PAGE and mass spectrometry data by helping to elucidate functional cellular pathways.