The traditional method of identifying a protein is to remove its amino acids one at a time and determine the identity of each one. This method is far too slow and labor-intensive for analyzing the thousands of proteins present in a typical cell. Today, researchers use mass spectrometry, which is a method for precisely determining the molecular mass of a molecule. In mass spectrometry, a molecule is ionized and its migration rate in an electrical field is determined. Because small molecules migrate more rapidly than do larger molecules, the migration rate can accurately determine the mass of the molecule.

To analyze a protein with mass spectrometry, researchers break it into small peptide fragments using a protein-digesting enzyme (Figure 15.13a). Mass spectrometry is then used to separate those peptides on the basis of their mass-to-charge (m/z) ratio (Figure 15.13b). This separation produces a profile of peaks, in which each peak corresponds to the mass-to-charge ratio of one peptide (Figure 15.13c). A computer program then searches through a database of proteins to find a match between the profile generated and the profile of a known protein (Figure 15.13d). Using bioinformatics, the computer creates “virtual digests” and predicts the profiles of all proteins found in a genome, given the DNA sequences of the protein-encoding genes.

Mass spectrometric methods can also be used to measure the amount of each protein identified. In this procedure, a complex mixture of proteins (such as those from a tissue sample) is digested and analyzed with mass spectrometry. The computer program then sorts out the proteins present in the sample by analyzing the peptide profiles.

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Mary Lipton and her colleagues used this approach to study the proteome of Deinococcus radiodurans, an exceptional bacterium that is able to withstand high doses of ionizing radiation that are lethal to all other organisms. The genome of D. radiodurans had already been sequenced. Lipton and her colleagues extracted proteins from the bacterium, broke them up into small peptide fragments, separated the fragments, and then used mass spectrometry to determine the proteins from the peptide fragments. They were able to identify 1910 proteins, which represented more than 60% of the proteins predicted on the basis of the genome sequence.