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Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity.

Kalb SR, Boyer AE, Barr JR - Toxins (Basel) (2015)

Bottom Line: One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum.There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases.Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum.

View Article: PubMed Central - PubMed

Affiliation: Centers for Disease Control and Prevention, 4770 Buford Hwy NE, Atlanta, GA 30341, USA. skalb@cdc.gov.

ABSTRACT
Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin.

No MeSH data available.


Related in: MedlinePlus

(B) MALDI-TOF mass spectrum indicating the presence of anthrax lethal factor; (A) Sequence of substrate and cleaved product and expected m/z of each. Peptide cleavage products indicating the presence of the anthrax lethal factor are marked with asterisks. X = norleucine.
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toxins-07-03497-f005: (B) MALDI-TOF mass spectrum indicating the presence of anthrax lethal factor; (A) Sequence of substrate and cleaved product and expected m/z of each. Peptide cleavage products indicating the presence of the anthrax lethal factor are marked with asterisks. X = norleucine.

Mentions: Another example of mass spectrometric detection of enzymatic activity of a protein toxin developed by our laboratory is for detection of anthrax lethal factor [45]. In this work, LF was extracted from serum using murine monoclonal antibodies to the toxin. The captured toxin was then added to a reaction buffer containing an optimized peptide substrate which has a similar amino acid sequence to the portion of MAPKK which is cleaved by anthrax lethal factor. In the presence of the toxin, the peptide substrate was cleaved and similar to the BoNT activity method, the peptide cleavage products were detected by MALDI-TOF mass spectrometry as shown in Figure 5. The intensity of the cleavage products is directly related to the amount of anthrax lethal factor; as the amount of anthrax lethal factor increases, the intensity of the cleavage products also increases in a linear fashion. MALDI-TOF MS quantification was possible through the addition of isotopically-labeled peptide cleavage products which have the same amino acid sequence as the peptide cleavage products yet have a higher mass due to incorporation of isotopically-labeled amino acid residues. These labeled peptide cleavage products were added equally to each sample to serve as an internal standard. Through comparison of the area of the peaks of the native and labeled peptide cleavage products, a determination of the amount of peptide cleavage product produced by LF can be made. Thus, the method for detection of anthrax lethal factor is one which is easily quantifiable, provided that the proper calibrants are used in the analysis.


Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity.

Kalb SR, Boyer AE, Barr JR - Toxins (Basel) (2015)

(B) MALDI-TOF mass spectrum indicating the presence of anthrax lethal factor; (A) Sequence of substrate and cleaved product and expected m/z of each. Peptide cleavage products indicating the presence of the anthrax lethal factor are marked with asterisks. X = norleucine.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591662&req=5

toxins-07-03497-f005: (B) MALDI-TOF mass spectrum indicating the presence of anthrax lethal factor; (A) Sequence of substrate and cleaved product and expected m/z of each. Peptide cleavage products indicating the presence of the anthrax lethal factor are marked with asterisks. X = norleucine.
Mentions: Another example of mass spectrometric detection of enzymatic activity of a protein toxin developed by our laboratory is for detection of anthrax lethal factor [45]. In this work, LF was extracted from serum using murine monoclonal antibodies to the toxin. The captured toxin was then added to a reaction buffer containing an optimized peptide substrate which has a similar amino acid sequence to the portion of MAPKK which is cleaved by anthrax lethal factor. In the presence of the toxin, the peptide substrate was cleaved and similar to the BoNT activity method, the peptide cleavage products were detected by MALDI-TOF mass spectrometry as shown in Figure 5. The intensity of the cleavage products is directly related to the amount of anthrax lethal factor; as the amount of anthrax lethal factor increases, the intensity of the cleavage products also increases in a linear fashion. MALDI-TOF MS quantification was possible through the addition of isotopically-labeled peptide cleavage products which have the same amino acid sequence as the peptide cleavage products yet have a higher mass due to incorporation of isotopically-labeled amino acid residues. These labeled peptide cleavage products were added equally to each sample to serve as an internal standard. Through comparison of the area of the peaks of the native and labeled peptide cleavage products, a determination of the amount of peptide cleavage product produced by LF can be made. Thus, the method for detection of anthrax lethal factor is one which is easily quantifiable, provided that the proper calibrants are used in the analysis.

Bottom Line: One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum.There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases.Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum.

View Article: PubMed Central - PubMed

Affiliation: Centers for Disease Control and Prevention, 4770 Buford Hwy NE, Atlanta, GA 30341, USA. skalb@cdc.gov.

ABSTRACT
Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin.

No MeSH data available.


Related in: MedlinePlus