{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,8]],"date-time":"2026-01-08T10:07:23Z","timestamp":1767866843955,"version":"3.49.0"},"reference-count":51,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2011,3,14]],"date-time":"2011-03-14T00:00:00Z","timestamp":1300060800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI) tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA) which combines with lethal factor (LF) and edema factor (EF), forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS\/MS. Pre-concentration\/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase.<\/jats:p>","DOI":"10.3390\/molecules16032391","type":"journal-article","created":{"date-parts":[[2011,3,14]],"date-time":"2011-03-14T14:24:25Z","timestamp":1300112665000},"page":"2391-2413","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":49,"title":["Quantitative Mass Spectrometry for Bacterial Protein Toxins \u2014 A Sensitive, Specific, High-Throughput Tool for Detection and Diagnosis"],"prefix":"10.3390","volume":"16","author":[{"given":"Anne E.","family":"Boyer","sequence":"first","affiliation":[{"name":"Centers for Disease Control and Prevention, 4770 Buford Hwy, NE, Atlanta, GA 30341, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maribel","family":"Gallegos-Candela","sequence":"additional","affiliation":[{"name":"Centers for Disease Control and Prevention, 4770 Buford Hwy, NE, Atlanta, GA 30341, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Renato C.","family":"Lins","sequence":"additional","affiliation":[{"name":"Battelle Analytical Services, Atlanta, at the Centers for Disease Control and Prevention, 4770 Buford Hwy, NE, Atlanta, GA 30341, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zsuzsanna","family":"Kuklenyik","sequence":"additional","affiliation":[{"name":"Centers for Disease Control and Prevention, 4770 Buford Hwy, NE, Atlanta, GA 30341, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Adrian","family":"Woolfitt","sequence":"additional","affiliation":[{"name":"Centers for Disease Control and Prevention, 4770 Buford Hwy, NE, Atlanta, GA 30341, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hercules","family":"Moura","sequence":"additional","affiliation":[{"name":"Centers for Disease Control and Prevention, 4770 Buford Hwy, NE, Atlanta, GA 30341, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Suzanne","family":"Kalb","sequence":"additional","affiliation":[{"name":"Centers for Disease Control and Prevention, 4770 Buford Hwy, NE, Atlanta, GA 30341, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Conrad P.","family":"Quinn","sequence":"additional","affiliation":[{"name":"Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA 30333, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"John R.","family":"Barr","sequence":"additional","affiliation":[{"name":"Centers for Disease Control and Prevention, 4770 Buford Hwy, NE, Atlanta, GA 30341, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2011,3,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1016\/S0009-8981(99)00228-4","article-title":"Bacterial toxins with intracellular protease activity","volume":"291","author":"Rossetto","year":"2000","journal-title":"Clin.Chim. Acta"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1111\/j.1462-5822.2006.00866.x","article-title":"Contribution of toxins to the pathogenesis of inhalational anthrax","volume":"9","author":"Tournier","year":"2007","journal-title":"Cell Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"3916","DOI":"10.1021\/ac050485f","article-title":"From the mouse to the mass spectrometer: detection and differentiation of the endoproteinase activities of botulinum neurotoxins A-G by mass spectrometry","volume":"77","author":"Boyer","year":"2005","journal-title":"Anal.Chem."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.ab.2006.01.027","article-title":"The use of Endopep-MS for the detection of botulinum toxins A, B, E, and F in serum and stool samples","volume":"351","author":"Kalb","year":"2006","journal-title":"Anal.Biochem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e5355","DOI":"10.1371\/journal.pone.0005355","article-title":"Extraction and inhibition of enzymatic activity of botulinum neurotoxins\/A1, \/A2, and \/A3 by a panel of monoclonal anti-BoNT\/A antibodies","volume":"4","author":"Kalb","year":"2009","journal-title":"PLoSOne"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3432","DOI":"10.1128\/IAI.00346-09","article-title":"Kinetics of lethal factor and poly-D-glutamic acid antigenemia during inhalation anthrax in rhesus macaques","volume":"77","author":"Boyer","year":"2009","journal-title":"Infect. Immun."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"8463","DOI":"10.1021\/ac701741s","article-title":"Detection and quantification of anthrax lethal factor in serum by mass spectrometry","volume":"79","author":"Boyer","year":"2007","journal-title":"Anal. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1098\/rstb.1999.0377","article-title":"Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses","volume":"354","author":"Pellizzari","year":"1999","journal-title":"Philos. Trans. R. Soc. Lond. B Biol. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1128\/JB.01180-06","article-title":"Genetic diversity among Botulinum Neurotoxin-producing clostridial strains","volume":"189","author":"Hill","year":"2007","journal-title":"J. Bacteriol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1152\/physrev.2000.80.2.717","article-title":"Neurotoxins affecting neuroexocytosis","volume":"80","author":"Schiavo","year":"2000","journal-title":"Physiol. Rev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/0167-4781(96)00006-1","article-title":"Mosaic structures of neurotoxins produced from Clostridium botulinum types C and D organisms","volume":"1307","author":"Moriishi","year":"1996","journal-title":"Biochim.Biophys. Acta"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Moura, H.T.R., Woolfitt, A.R., Gallegos-Candela, M., McWilliams, L.G., Solano, M.I., Pirkle, J.L., and Barr, J.R. (2011). Studies on Botulinum Neurotoxins Type \/C1 and mosaic \/DC Using Endopep-MS and Proteomics. FEMS Immunol. Microbiol., in press.","DOI":"10.1111\/j.1574-695X.2010.00774.x"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"5450","DOI":"10.1128\/IAI.73.9.5450-5457.2005","article-title":"Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization","volume":"73","author":"Smith","year":"2005","journal-title":"Infect. Immun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1046\/j.1462-5822.2003.00253.x","article-title":"Anthrax toxins and the host: A story of intimacy","volume":"5","author":"Mock","year":"2003","journal-title":"Cell Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1042\/BJ20061891","article-title":"Manipulation of host signalling pathways by anthrax toxins","volume":"402","author":"Turk","year":"2007","journal-title":"Biochem.J."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"10277","DOI":"10.1073\/pnas.89.21.10277","article-title":"Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin","volume":"89","author":"Klimpel","year":"1992","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"16396","DOI":"10.1016\/S0021-9258(18)42016-9","article-title":"Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen","volume":"267","author":"Molloy","year":"1992","journal-title":"J. Biol. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"614","DOI":"10.1016\/j.jmb.2009.07.037","article-title":"The protective antigen component of anthrax toxin forms functional octameric complexes","volume":"392","author":"Kintzer","year":"2009","journal-title":"J. Mol. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1871","DOI":"10.1016\/S0002-9440(10)62496-0","article-title":"Anthrax lethal toxin induces endothelial barrier dysfunction","volume":"166","author":"Warfel","year":"2005","journal-title":"Am. J. Pathol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1885","DOI":"10.1086\/657408","article-title":"Anthrax lethal and edema toxins produce different patterns of cardiovascular and renal dysfunction and synergistically decrease survival in canines","volume":"202","author":"Sweeney","year":"2010","journal-title":"J. Infect. Dis."},{"key":"ref_21","unstructured":"Boyer, A.E., Quinn, C.P., Hoffmaster, A.R., Kozel, T., Gallegos, M., Saile, E., Marston, C., Beesley, C., Percival, A., Lins, R., Woolfitt, A., Pirkle, J.L., and Barr, J.R. (2010, January 6-9). Association of Early Lethal Factor Levels with Survival during Inhalation Anthrax in Rhesus Macaques. Proceedings of ASM Biodefense and Emerging Diseases Research Meeting, Baltimore, MD, USA."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"968","DOI":"10.1086\/512372","article-title":"A case of naturally acquired inhalation anthrax: Clinical care and analyses of anti-protective antigen immunoglobulin G and lethal factor","volume":"44","author":"Walsh","year":"2007","journal-title":"Clin.Infect. Dis."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1093\/jaoac\/86.2.314","article-title":"Detection of botulinal neurotoxins A, B, E, and F by amplified enzyme-linked immunosorbent assay: Collaborative study","volume":"86","author":"Ferreira","year":"2003","journal-title":"J. AOAC Int."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"543","DOI":"10.1099\/00221287-138-3-543","article-title":"Serum protease cleavage of Bacillus anthracis protective antigen","volume":"138","author":"Ezzell","year":"1992","journal-title":"J. Gen. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"10834","DOI":"10.1074\/jbc.M412210200","article-title":"Purified Bacillus anthracis lethal toxin complex formed in vitro and during infection exhibits functional and biological activity","volume":"280","author":"Panchal","year":"2005","journal-title":"J. Biol. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1676","DOI":"10.1093\/clinchem\/42.10.1676","article-title":"Isotope dilution--mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I","volume":"42","author":"Barr","year":"1996","journal-title":"Clin. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2510","DOI":"10.1016\/j.vaccine.2008.03.014","article-title":"Quantification of influenza virus hemagglutinins in complex mixtures using isotope dilution tandem mass spectrometry","volume":"26","author":"Williams","year":"2008","journal-title":"Vaccine"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Kuklenyik, Z., Boyer, A.E., Lins, R., Quinn, C.P., Gallegos-Candela, M., Woolfitt, A.R., Pirkle, J.L., and Barr, J.R. (2011). Comparison of MALDI-TOF-MS and HPLC-ESI-MS\/MS for Endopeptidase Activity Based Quantification of Anthrax Lethal Factor in Serum. Anal. Chem., in press.","DOI":"10.1021\/ac1030144"},{"key":"ref_29","unstructured":"Lins, R.C., Boyer, A.E., Kuklenyik, Z., Quinn, C.P., Gallegos-Candela, M., Leysath, C.E., Chen, Z., Chen, K., Makiya, M., Leppla, S.H., Pirkle, J.L., and Barr, J.R. (2010, January 23-27). A High Sensitivity Adenylate Cyclase Assay for Anthrax Edema factor.. Proceedings of 58th ASMS Conference on Mass Spectrometry and Allied Topics, Salt Lake City, UT, USA."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1038\/nsmb708","article-title":"The structural basis for substrate and inhibitor selectivity of the anthrax lethal factor","volume":"11","author":"Turk","year":"2004","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1016\/S0014-5793(99)01502-1","article-title":"Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS\/IFNgamma-induced release of NO and TNFalpha","volume":"462","author":"Pellizzari","year":"1999","journal-title":"FEBS Lett."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"734","DOI":"10.1126\/science.280.5364.734","article-title":"Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor","volume":"280","author":"Duesbery","year":"1998","journal-title":"Science"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1042\/bj3520739","article-title":"Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor","volume":"352 (Pt. 3)","author":"Vitale","year":"2000","journal-title":"Biochem. J."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"29427","DOI":"10.1074\/jbc.M402689200","article-title":"Structural and kinetic analyses of the interaction of anthrax adenylylcyclase toxin with reaction products cAMP and pyrophosphate","volume":"279","author":"Guo","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1128\/CMR.18.2.247-263.2005","article-title":"Clostridium difficile toxins: Mechanism of action and role in disease","volume":"18","author":"Voth","year":"2005","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"15R","DOI":"10.1093\/glycob\/cwm004","article-title":"Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function","volume":"17","author":"Jank","year":"2007","journal-title":"Glycobiology"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3162","DOI":"10.1073\/pnas.79.10.3162","article-title":"Anthrax toxin edema factor: A bacterial adenylatecyclase that increases cyclic AMP concentrations of eukaryotic cells","volume":"79","author":"Leppla","year":"1982","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_38","unstructured":"Gallegos-Candela, M., Boyer, A.E., Quinn, C.P., Woolfitt, A.R., and Barr, J.R. (September,, January 30). Validation of Rapid, Sensitive, Quantitative MALDI-TOF MS Assays for Anthrax Lethal Factor and Lethal Toxin, Bacillus-ACT 2009. Proceedings of the International Bacillus Anthracis, B. Cereus, and B. Thuringiensis Conference, Santa Fe, NM, USA."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"5871","DOI":"10.1128\/IAI.00792-06","article-title":"Protective antigen as a correlative marker for anthrax in animal models","volume":"74","author":"Kobiler","year":"2006","journal-title":"Infect. Immun."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1128\/CVI.00023-06","article-title":"Detection of anthrax toxin in the serum of animals infected with Bacillus anthracis by using engineered immunoassays","volume":"13","author":"Mabry","year":"2006","journal-title":"Clin.Vaccine Immunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"e12237","DOI":"10.1371\/journal.pone.0012237","article-title":"Extraction of BoNT\/A, \/B, \/E, and \/F with a single, high affinity monoclonal antibody for detection of botulinum neurotoxin by Endopep-MS","volume":"5","author":"Kalb","year":"2010","journal-title":"PLoSOne"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"408","DOI":"10.1128\/CVI.00412-08","article-title":"Detection of anthrax toxin by an ultrasensitive immunoassay using europium nanoparticles","volume":"16","author":"Tang","year":"2009","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_43","unstructured":"Boyer, A.E., Quinn, C.P., Hoffmaster, A., Gallegos, M., Saile, E., Marston, C., Kozel, T., Meister, G., Woolfitt, A.R., Pirkle, J.L., and Barr, J.R. (2008, January 24-27). Kinetics of Toxemia of Inhalation Anthrax. In. Proceedings of 6th Annual ASM Biodefense and Emerging Diseases Research Meeting, Baltimore, MD, USA."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"6140","DOI":"10.1021\/ac0511748","article-title":"Detection of botulinum neurotoxin A in a spiked milk sample with subtype identification through toxin proteomics","volume":"77","author":"Kalb","year":"2005","journal-title":"Anal. Chem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/j.jchas.2008.03.013","article-title":"Biological Monitoring of Exposure to Botulinum Neurotoxins","volume":"15","author":"Barr","year":"2008","journal-title":"Chem. Health Safety"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"897","DOI":"10.1586\/14787210.6.6.897","article-title":"Measuring the severity of Clostridium difficile infection: implications for management and drug development","volume":"6","author":"Belmares","year":"2008","journal-title":"Expert Rev. Anti Infect. Ther."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1185\/030079908X253735","article-title":"Do fluoroquinolones predispose patients to Clostridium difficile associated disease? A review of the evidence","volume":"24","author":"Deshpande","year":"2008","journal-title":"Curr.Med. Res. Opin."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1230","DOI":"10.1086\/514776","article-title":"Changing epidemiology of pertussis in the United States: increasing reported incidence among adolescents and adults, 1990-1996","volume":"28","author":"Guris","year":"1999","journal-title":"Clin. Infect. Dis."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1186\/1471-2164-9-311","article-title":"Comparative genomic profiling of Dutch clinical Bordetellapertussis isolates using DNA microarrays: Identification of genes absent from epidemic strains","volume":"9","author":"King","year":"2008","journal-title":"BMC Genomics"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"757","DOI":"10.1586\/14760584.4.5.757","article-title":"Are vaccination programs and isolate polymorphism linked to pertussis re-emergence?","volume":"4","author":"Godfroid","year":"2005","journal-title":"Expert Rev. Vaccines"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1301","DOI":"10.1128\/AEM.01662-10","article-title":"Clostridium baratii Type F Neurotoxin Cleaves Synaptobrevin-2 with Different Substrate Recognition Requirement than Clostridium botulinum Type F Neurotoxin","volume":"77","author":"Kalb","year":"2011","journal-title":"Appl. Environ. Microbiol."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/16\/3\/2391\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:55:31Z","timestamp":1760219731000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/16\/3\/2391"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2011,3,14]]},"references-count":51,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2011,3]]}},"alternative-id":["molecules16032391"],"URL":"https:\/\/doi.org\/10.3390\/molecules16032391","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2011,3,14]]}}}