Abstract
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.
Highlights
Bacterial protein toxins are among the most potent natural poisons known. Members of this group of bioactive polypeptides can cause irreversible changes to host cellular targets resulting in a wide variety of functional losses, ranging from the paralyses caused by clostridial neurotoxins, to the immune collapse, endothelial dysfunction, hemorrhage and death due to the anthrax toxins [1,2]
Enrichment using monoclonal antibodies specific for the toxin that are covalently bound to magnetic beads, 2) toxin-specific enzyme activity directed against a specific substrate, and 3) mass specific detection of the toxin-generated reaction products
We found that detection limits, accuracy and precision were very similar between the two methods
Summary
Bacterial protein toxins are among the most potent natural poisons known. Members of this group of bioactive polypeptides can cause irreversible changes to host cellular targets resulting in a wide variety of functional losses, ranging from the paralyses caused by clostridial neurotoxins (flaccid paralysis or tonic spasms), to the immune collapse, endothelial dysfunction, hemorrhage and death due to the anthrax toxins [1,2]. Rather than detecting molecular levels of a substance, protein, or enzyme toxin in the blood, detecting the toxins activity in the presence of excess substrate allows accumulation of the enzyme-specific reaction products over time. Analytical MS-based detection is one of the important features of these toxin activity/reaction product targeted methods. Enrichment using monoclonal antibodies (mAbs) specific for the toxin that are covalently bound to magnetic beads, 2) toxin-specific enzyme activity directed against a specific substrate, and 3) mass specific detection of the toxin-generated reaction products. This method schematic is shown for MS detection of the anthrax toxins, LF and EF (Figure 2). ATP and the adenylyl cyclase cofactor calmodulin, ATP and the adenylyl cyclase reaction product cAMP are detected by LC-ESI-MS/MS
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