Stable Isotope Labeling with Amino Acids as an Aid to Protein Identification in Peptide Mass Fingerprinting

Peptide mass fingerprinting is arguably the simplest one-dimensional method used in proteomics to identify proteins, whether recovered from bands or spots on one-dimensional (1-D) or two-dimensional (2-D) gels, as single proteins or mixtures isolated by chromatographic steps, or by affinity purification. In all instances, the protein to be identified is first isolated and then digested with a protease of defined specificity, such as trypsin or endopeptidase LysC. These peptides are then mass measured, usually by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Because the protein was isolated prior to proteolytic fragmentation, connectivity between the product peptides can reasonably be inferred and the resulting peptide mass fingerprint can be used to search protein databases (1,2).