Protein Identification and Characterization through Peptide Mass Spectrometry Method Development for Improved Ricin and Botulinum Neurotoxin Analysis

The illicit usage of toxic substances is increasing rapidly around the world, creating a need for comprehensive chemical methods for detecting and quantitating toxic agents that might be used in criminal activities or terror attacks. There are several toxic proteins that could potentially be used in bioterrorism or biocrime. For example, the plant toxins ricin and abrin are readily available, very toxic, and easily produced, and are therefore frequently involved in biocrime incidents. Another threat agent is the extremely poisonous botulinum neurotoxin, which is among the most toxic substances known. Forensic analyses of samples potentially containing these diverse and very toxic agents therefore require analytical methods capable of detecting trace amounts of the target analyte in complex mixtures. This thesis describes the development of new methods for peptide mass spectrometry that offer improved performance in forensic toxin analysis. A galactose affinity method was developed for the enrichment and subsequent analysis of ricin, abrin and botulinum neurotoxin. The method’s applicability was confirmed during a forensic investigation into illegal toxin preparations. Additionally, the investigations generated new information on the structural properties of ricin that will facilitate the forensic matching of samples to sources. Details of botulinum neurotoxin’s sophisticated intoxication mechanisms were determined by using a broad analytical approach to study the importance and heterogeneity of SV2C glycosylation. Overall, the results and procedures presented in this thesis will help to improve analytical capabilities relevant to the detection and prevention of biocrime and bioterrorism. More generally, it provides methodological guidance and useful strategies for researchers in peptide mass spectrometry.