Mass Spectrometry

Mass spectrometry is a powerful tool for accurately detecting and identifying specific proteins separated by 2-dimensional (2D) electrophoresis. First evolving in the early 1990s, the core technique has essentially three steps. In the first step of mass spectrometry, a complex protein sample is separated by 2D electrophoresis. By this method, the protein sample (not treated with SDS) is loaded into one well of a polyacrylamide gel that possesses a pH gradient, usually from 3-10, within the gel matrix. When the proteins in the complex sample travel through the gel during electrophoresis, they stop migrating at the pH at which the net electric charge of the molecule is zero. This pH value is called the isoelectric point or "pI" (1st dimension). After the proteins in the sample are separated according to their pI value, the entire lane of the gel is removed and laid across the top of a new, normal polyacrylamide gel (e.g., without a pH gradient), and the proteins are then further separated by their molecular weight (i.e., size; 2nd dimension) during a second electrophoresis step. During the second step of mass spectrometry, protein bands of interest are then excised from the second gel. Then the gel plug containing the protein-of-inter-est is digested using the enzyme trypsin. In the third step of the procedure, the peptides that result from the trypsin digest of the protein-of-interest are analyzed by mass spectrom-etry and the spectra produced by the collision-induced dissociation is searched against mass spectra of peptide sequences contained in a database.

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