Bacterial and mammalian cells are rich in putrescine, spermidine and spermine. Polyamines can be incorporated into proteins in vitro. Very few naturally occurring polyaminated proteins have been identified. Bovine albumin and the recombinant universal stress protein from Francisella tularensis were used as models for mass spectrometry analysis of polyaminated proteins. The proteins were covalently bound to putrescine, spermidine, or spermine by the action of carbodiimide or microbial transglutaminase. Tryptic peptides were subjected to liquid chromatography tandem mass spectrometry. Adducts were identified by Protein Prospector software. We describe the search parameters for identifying polyaminated peptides in Protein Prospector and show convincing MMS spectra for adducts with putrescine, spermidine, and spermine. Manual evaluation led us to recognize signature ions for polyamine adducts on Asp, Glu, and Gln. Manual evaluation recognized neutral loss from putrescine, spermidine and spermine during the fragmentation process. Mechanisms for formation of signature ions and neutral loss are presented. Manual evaluation recognized a false positive adduct which had been formed during trypsinolysis by rearrangement of a peptide sequence. Another false positive was a 71 Da added mass on cysteine, which was initially assumed to be putrescine, but was actually a propionamide adduct for a sample extracted from a polyacrylamide gel. The type of information presented in this report serves as a model for identifying naturally occurring polyaminated proteins.