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Clostridium botulinum produces botulinum neurotoxins (BoNTs) that are one of the most poisonous substances. In order to respond to public health emergencies, there is a need to develop sensitive and specific methods for detecting botulinum toxin in various clinical matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate BoNT serotypes A-G by immunoaffinity capture of toxins and detection of unique cleavage products of peptide substrates. To improve the sensitivity of the Endopep-MS assay for the detection of BoNT serotype G, we report here the optimization of synthetic peptide substrates through systematic substitution, deletion, and incorporation of unnatural amino acids. Our data show that the resulting optimized peptides produced a significant improvement (two orders of magnitude) in assay sensitivity and allowed the detection of 0.01 mouseLD 50 toxin present in buffer solution.

Rapid and sensitive detection of botulinum neurotoxins (BoNTs), which cause botulism, is essential in a public health emergency or bioterrorism event. We have previously developed a mass spectrometry (MS)-based functional method, Endopep-MS assay, for the fast detection and differentiation of all BoNT serotypes by affinity enriching the toxin and detecting the serotype-specific cleavage products of peptide substrates derived from the in vivo targets. To improve the performance of the Endopep-MS assay, we report here the further optimization of the peptide substrate for the detection of serotype A botulinum neurotoxins. An increased substrate cleavage was achieved by extending the original peptide N-terminus with optimized amino acid sequence, increasing the detection sensitivity of the method. In addition, the resistance of the substrate to nonspecific hydrolysis was dramatically improved by selectively substituting amino acids at the scissile bond and various other positions of the extended peptide. Moreover, incorporating the N-terminal hydrophobic residues dramatically improved the relative intensity of the cleavage products in the mass spectra. This allowed easy detection of the cleavage products, further enhancing the performance of the assay. The limit of detection for spiked serum sample was enhanced from 0.5 to 0.1 mouseLD 50 and from 0.5 to 0.2 mouseLD 50 for spiked stool. Graphical abstract Mass spectra of optimized and old peptide substrates with BoNT/A.

It is essential to have a simple, quick and sensitive method for the detection and quantification of botulinum neurotoxins, the most toxic substances and the causative agents of botulism. Type C botulinum neurotoxin (BoNT/C) represents one of the seven members of distinctive BoNT serotypes (A to G) that cause botulism in animals and avians. Here we report the development of optimized peptide substrates for improving the detection of BoNT/C and /CD mosaic toxins using an Endopep-MS assay, a mass spectrometry-based method that is able to rapidly and sensitively detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Based on the sequence of a short SNAP-25 peptide, we conducted optimization through a comprehensive process including length determination, terminal modification, single and multiple amino acid residue substitution, and incorporation of unnatural amino acid residues. Our data demonstrate that an optimal peptide provides a more than 200-fold improvement over the substrate currently used in the Endopep-MS assay for the detection of BoNT/C1 and /CD mosaic. Using the new substrate in a four-hour cleavage reaction, the limit of detection for the BoNT/C1 complex spiked in buffer, serum and milk samples was determined to be 0.5, 0.5 and 1mouseLD50/mL, respectively, representing a similar or higher sensitivity than that obtained by traditional mouse bioassay., (Published by Elsevier Ltd.)

N-terminal derivatization of peptides with Edman's reagent, phenyl isothiocyanate (PITC), promotes gas-phase Edman cleavage that yields abundant complementary b(1) and y(n-1) ion pairs by tandem mass spectrometry (MS/MS). The formation of b(1) ions can be utilized as a mass tag to enhance the interpretation of MS/MS spectra and increase the confidence of peptide identification during mass spectrometry analysis. Derivatization of tryptic peptides with another isothiocyanate analogue, 4-sulfophenyl isothiocyanate, also produces signature ions resulting from Edman cleavage and facilitates peptide sequencing on linear or branched peptides. The limitation of these derivatizations, however, is reduced MS signal intensities of modified peptides, due presumably to the tags themselves. Here we have demonstrated that several other isothiocyanate analogues bearing basic moieties can derivatize peptides and significantly improve the MS sensitivity of tagged analytes, while promoting Edman fragmentation and maintaining other sequence fragments as well.

Post source decay (PSD) analysis of precursor ions generated from matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is a powerful tool for amino acid sequencing and primary structure analysis of proteins. N-Terminal sulfonation has become an effective derivatization strategy in facilitating de novo peptide sequencing by the formation of predominate y-type ion series in MALDI PSD spectra. Recently, an effective and inexpensive N-terminal derivatization method has been reported using 4-sulfophenyl isothiocyanate (SPITC) as the derivatization reagent (J. Mass. Spectrom. 2003; 38: 373-377). In this paper, we report an improvement in the derivatization procedure with this reagent that involves replacing an organic co-reagent with other chemicals and eliminating the use of organic solvent. The method is demonstrated on a model peptide and on tryptic digests of two proteins. The results indicate that the improved sulfonation reaction can be implemented with high efficiency under aqueous conditions and that the sensitivity of mass detection can be increased considerably., (Copyright 2003 John Wiley & Sons, Ltd.)

Abstract: Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to humans. Intoxication with BoNT causes flaccid paralysis and can lead to death if untreated with serotype-specific antibodies. Supportive care, including ventilation, may be necessary. Rapid and sensitive detection of BoNT is necessary for timely clinical confirmation of clinical botulism. Previously, our laboratory developed a fast and sensitive mass spectrometry (MS) method termed the Endopep–MS assay. The BoNT serotypes are rapidly detected and differentiated by extracting the toxin with serotype-specific antibodies and detecting the unique and serotype-specific cleavage products of peptide substrates that mimic the sequence of the BoNT native targets. To further improve the sensitivity of the Endopep–MS assay, we report here the optimization of the substrate peptide for the detection of BoNT/A. Modifications on the terminal groups of the original peptide substrate with acetylation and amidation significantly improved the detection of BoNT/A cleavage products. The replacement of some internal amino acid residues with single or multiple substitutions led to further improvement. An optimized peptide increased assay sensitivity 5-fold with toxin spiked into buffer solution or different biological matrices. [Copyright &y& Elsevier]

Protein ubiquitination plays an important role in the degradation and other functional regulation of cellular proteins in organisms ranging from yeasts to mammals. Trypsin digestion of ubiquitin conjugated proteins produces diglycine branched peptides in which the C-terminal Gly-Gly fragment of ubiquitin is attached to the ϵ-amino group of a modified lysine residue within the peptide. This provides a platform for mapping ubiquitination sites using mass spectrometry. Here we report the development of a novel strategy for determining posttraslational protein ubiquitination based on the N-terminal sulfonation of diglycine branched peptides. In contrast to conventional tandem MS spectra of native tryptic peptides, MALDI MS/MS analysis of a sulfonated tryptic peptide containing a diglycine branch generates a unique spectrum composed of a signature portion and a sequence portion. The signature portion of the spectrum consists of several intense ions resulting from the elimination of the tags, the N-terminal residues at the peptide and the branch, and their combination. This unique ion distribution pattern can distinguish ubiquitination modificatons from others and can identify the first N-terminal residues of the peptides as well. The sequence portion consists of an exclusive series of y-type ions and y' ions (differing by the loss of one glycine residue from the sulfonated diglycine branch) that can directly reveal the amino acid sequence of the peptide and the precise location of the ubiquitination site. The technique is demonstrated for a series of synthetic peptides and is validated by a model protein, tetraubiquitin. Our results show that the MALDI MS/MS analysis of sulfonated tryptic peptides can provide a highly effective method for the determination of ubiquitination substrates, ubiquitination sites on protein targets, and modification sites on ubiquitins themselves. [ABSTRACT FROM AUTHOR]