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Recombinant protective antigen (rPA) contains similar isoforms that appear early in fermentation and are mostly removed through purification. By liquid chromatography\u2010tandem mass spectrometry sequencing of the entire protein and inspection of spectral data for amino acid modifications, pharmaceutical rPA contained measurable deamidation at seven of its 68 asparagine residues. A direct association between isoform complexity and percent deamidation was observed such that each decreased with purity and increased with protein aging. Position N537 consistently showed the highest level of modification, although its predicted rate of deamidation ranked 10th by theoretical calculation, and other asparagines of higher predicted rates were observed to be unmodified. rPA with more isoforms and greater deamidation displayed lower activities for furin cleavage, heptamerization, and holotoxin formation. Lethal factor\u2010mediated macrophage toxicity correlated inversely with deamidation at residues N466 and N408. The described method measures deamidation without employing theoretical isotopic distributions, comparison between differentially treated samples or computational predictions of reactivity rates, and is broadly applicable to the characterization of other deamidated proteins. Proteins 2007. \u00a9 2007 Wiley\u2010Liss, Inc.<\/jats:p>","DOI":"10.1002\/prot.21432","type":"journal-article","created":{"date-parts":[[2007,4,27]],"date-time":"2007-04-27T21:48:36Z","timestamp":1177710516000},"page":"458-479","source":"Crossref","is-referenced-by-count":25,"title":["Multiple asparagine deamidation of <i>Bacillus anthracis<\/i> protective antigen causes charge isoforms whose complexity correlates with reduced biological activity"],"prefix":"10.1002","volume":"68","author":[{"given":"Bradford S.","family":"Powell","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeffrey T.","family":"Enama","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wilson 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