Detection and Quantification of Anthrax Lethal Factor in Serum by Mass Spectrometry

• Published in: Analytical chemistry (Washington) 2007-11, Vol.79 (22), p.8463-8470
• Main Authors: Boyer, Anne E, Quinn, Conrad P, Woolfitt, Adrian R, Pirkle, James L, McWilliams, Lisa G, Stamey, Karen L, Bagarozzi, Dennis A, Hart, John C, Barr, John R
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical chemistry; Animals; Anthrax; Antibodies - immunology; Antigens; Antigens, Bacterial - blood; Antigens, Bacterial - chemistry; Antigens, Bacterial - immunology; Bacillus anthracis; Bacteria; Bacterial Toxins - blood; Bacterial Toxins - chemistry; Bacterial Toxins - immunology; Chemistry; Exact sciences and technology; Kinases; Macaca mulatta; Macaca mulatta - blood; Mass spectrometry; Mitogen-Activated Protein Kinase Kinases - chemistry; Mitogen-Activated Protein Kinase Kinases - classification; Molecular Sequence Data; Peptides - chemistry; Peptides - metabolism; Sequence Alignment; Spectrometric and optical methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Substrate Specificity; T cell receptors; Toxins
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac701741s

The lethal toxin produced during Bacillus anthracis infection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF), a zinc-dependent endoproteinase whose known targets include five members of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators. We have developed a method for detecting functional LF in serum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to capture and concentrate the LF from serum. The captured LF was exposed to an optimized MAPKK-based peptide substrate, which it hydrolyzed into two smaller peptides. The LF cleavage products were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. The entire analytical method can be performed in less than 4 h with detection of LF levels as low as 0.05 ng/mL. The method was used to quantify LF levels in serum from rhesus macaques infected with B. anthracis. Serum samples obtained at day 2 postinfection contained 30−250 ng/mL LF and illustrated the clear potential to detect LF earlier in the infection cycle. This method represents a highly specific and rapid diagnostic tool for early anthrax and has a potential additional role as a research tool for understanding toxemia and effects of medical countermeasures for anthrax.