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•Mass spectrometry-based proteomics is well established in the fundamental life sciences.•Proteomics has many current emerging applications biological forensics and related areas.•Applications include both targeted and untargeted (discovery) methods.•Advances in peptide identification and sequence database selection will result in improvements. Mass spectrometry-based proteomics has been a useful tool for addressing numerous questions in basic biology research for many years. This success, combined with the maturity of mass spectrometric instrumentation, the ever-increasing availability of protein sequence databases derived from genome sequencing, and the growing sophistication of data analysis methods, places proteomics in a position to have an important role in biological forensics. Because proteins contain information about genotype (sequence) and phenotype (expression levels), proteomics methods can both identify biological samples and characterize the conditions that produced them. In addition to serving as a valuable orthogonal method to genomic analyses, proteomics can be used in cases where nucleic acids are absent, degraded, or uninformative. Mass spectrometry provides both broad applicability and exquisite specificity, often without customized detection reagents like primers or antibodies. This review briefly introduces proteomics methods, and surveys a variety of forensic applications (including criminal justice, historical, archaeological, and national security areas). Finally, challenges and crucial areas for further research are addressed.

The spore forming pathogen Bacillus anthracis is the etiologic agent of anthrax in humans and animals. It cycles through infected hosts as vegetative cells and is eventually introduced into the environment where it generates an endospore resistant to many harsh conditions. The endospores are subsequently taken up by another host to begin the next cycle. Outbreaks of anthrax occur regularly worldwide in wildlife and livestock, and the potential for human infection exists whenever humans encounter infected animals. It is also possible to encounter intentional releases of anthrax spores, as was the case in October 2001. Consequently, it is important to be able to rapidly establish the provenance of infectious strains of B. anthracis. Here, we compare protein expression in seven low-passage wild isolates and four laboratory strains of B. anthracis grown under identical conditions using LC-MS/MS proteomic analysis. Of the 1,023 total identified proteins, 96 had significant abundance differences between wild and laboratory strains. Of those, 28 proteins directly related to sporulation were upregulated in wild isolates, with expression driven by Spo0A, CodY, and AbrB/ScoC. In addition, we observed evidence of changes in cell division and fatty acid biosynthesis between the two classes of strains, despite being grown under identical experimental conditions. These results suggest wild B. anthracis cells are more highly tuned to sporulate than their laboratory cousins, and this difference should be exploited as a method to differentiate between laboratory and low passage wild strains isolated during an anthrax outbreak. This knowledge should distinguish between intentional releases and exposure to strains in nature, providing a basis for the type of response by public health officials and investigators.

Antimicrobial resistance (AMR) is a well-recognized, widespread, and growing issue of concern. With increasing incidence of AMR, the ability to respond quickly to infection with or exposure to an AMR pathogen is critical. Approaches that could accurately and more quickly identify whether a pathogen is AMR also are needed to more rapidly respond to existing and emerging biological threats. We examined proteins associated with paired AMR and antimicrobial susceptible (AMS) strains of Yersinia pestis and Francisella tularensis , causative agents of the diseases plague and tularemia, respectively, to identify whether potential existed to use proteins as signatures of AMR. We found that protein expression was significantly impacted by AMR status. Antimicrobial resistance-conferring proteins were expressed even in the absence of antibiotics in growth media, and the abundance of 10–20% of cellular proteins beyond those that directly confer AMR also were significantly changed in both Y. pestis and F. tularensis . Most strikingly, the abundance of proteins involved in specific metabolic pathways and biological functions was altered in all AMR strains examined, independent of species, resistance mechanism, and affected cellular antimicrobial target. We have identified features that distinguish between AMR and AMS strains, including a subset of features shared across species with different resistance mechanisms, which suggest shared biological signatures of resistance. These features could form the basis of novel approaches to identify AMR phenotypes in unknown strains.

In previous work, we demonstrated the use of electrospray ionization to analyze small differences in size or sequence of relatively small polymerase chain reaction (PCR) products of 114 base pairs or less. The sequence information required to answer a biological question may be only a single nucleotide substitution or deletion. In many cases, the regions where these sequence variations can occur are several hundred base pairs in length, and the analysis of large PCR products is therefore desirable. Therefore, we have attempted to expand the size range of PCR products that can be analyzed by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Previous work has shown that the difficulties associated with PCR product analysis increase with product size. A revised cleanup scheme was employed to target the removal of detergents with ethanol wash or precipitation steps, followed by additional desalting. Additionally, an in-trap cleanup to collisionally induce dissociation of noncovalent salt adducts was employed. This approach was extended to a 223 base pair PCR product yielding mass measurement accuracy within 26 ppM. The mass measurement accuracy obtained illustrates that a single base substitution could be identified at this size of PCR product with a 7 tesla ESI-FTICR.

In previous work, we demonstrated the use of electrospray ionization to analyze small differences in size or sequence of relatively small polymerase chain reaction (PCR) products of 114 base pairs or less. The sequence information required to answer a biological question may be only a single nucleotide substitution or deletion. In many cases, the regions where these sequence variations can occur are several hundred base pairs in length, and the analysis of large PCR products is therefore desirable. Therefore, we have attempted to expand the size range of PCR products that can be analyzed by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Previous work has shown that the difficulties associated with PCR product analysis increase with product size. A revised cleanup scheme was employed to target the removal of detergents with ethanol wash or precipitation steps, followed by additional desalting. Additionally, an in-trap cleanup to collisionally induce dissociation of noncovalent salt adducts was employed. This approach was extended to a 223 base pair PCR product yielding mass measurement accuracy within 26 ppM. The mass measurement accuracy obtained illustrates that a single base substitution could be identified at this size of PCR product with a 7 tesla ESI-FTICR.

The identification of biosignatures of aerosol exposure to pathogens has the potential to provide useful diagnostic information. In particular, markers of exposure to different types of respiratory pathogens may yield diverse sets of markers that can be used to differentiate exposure. We examine a mouse model of aerosol exposure to known Gram negative bacterial pathogens, Francisella tularensis novicida and Pseudomonas aeruginosa. Mice were subjected to either a pathogen or control exposure and bronchial alveolar lavage fluid (BALF) was collected at four and twenty four hours post exposure. Small protein and peptide markers within the BALF were detected by matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and analyzed using both exploratory and predictive data analysis methods; principle component analysis and degree of association. The markers detected were successfully used to accurately identify the four hour exposed samples from the control samples. This report demonstrates the potential for small protein and peptide marker profiles to identify aerosol exposure in a short post-exposure time frame.

We have attempted to expand the size range of PCR products that can be analyzed by electroscopy ionization (ESI) Fourier transformion cyclotron resonance (FTICR) mass spectrometry. The mass measurement accuracy obtained illustrates that a signel base substitution could be identified at the size of PCR product with a 7 tesla ESI-FTICR

The detection of high-energy γ-ray sources is vitally important to national security for numerous reasons, particularly nuclear materials smuggling interdiction and threat detection. This article presents two methods of detecting and locating a concealed nuclear γ-ray source by analyzing detector data of emissions that have been modulated with a coded mask. The advantages of each method, derived from a simulation study and experimental data, are discussed. Energetic γ-rays readily penetrate moderate amounts of shielding material and can be detected at distances of many meters. Coded masks are spatial configurations of shielding material (e.g., small squares formed from plates of lead or tungsten) placed in front of a detector array to modulate the radiation distribution. A coded mask system provides improved detection through an increased signal-to-noise ratio. In a search scenario it is impossible to obtain a comparison background run without the presence of a potential concealed source. The developed analysis methods simultaneously estimate background and source emissions and thus provide the capability to detect and locate a concealed high-energy radiological source in near real time. An accurate source location estimate is critically important to expedite the investigation of a high-probability γ-ray source. The experimental examples presented use a proof-of-concept coded mask system of a 4 × 4 array of NaI detectors directed at a γ-ray source in a field-of-view roughly 4 m wide × 3 m high (approximately the size of the side panel of a small freight truck). Test results demonstrate that the correct location of a radiologic source could be determined in as little as 100 seconds when the source was 6 m from the detector.

Differentiation between members of the B. cereus group (B. cereus, B. anthracis and B. thuringiensis) is a difficult taxonomic problem because of their high degree of phenotypic and genotypic similarity. Therefore this group was the focus of a taxonomic study using carbohydrate profiling and comparison of common genomic regions. B. subtilis was included as a more distantly related Bacillus. B. thuringiensis was found to be quite similar to B. cereus in carbohydrate composition for both vegetative and spore forms. A change in carbohydrate composition on transition from vegetative cell to spore was noted for the B. cereus group and B. subtilis. It was unclear whether the carbohydrate transition observed on sporulation was related to the teichoic/teichuronic acid transition (described by Ellwood and Tempest) where bacilli change their carbohydrate composition when grown under conditions of phosphate deprivation. An existing gas chromatography mass spectrometry procedure was available for studying neutral and amino sugar components of these cells. However, in order to analyze glucuronic acid (an acidic sugar marker for teichuronic acid) it was necessary to develop a liquid chromatography electrospray ionization tandem mass spectrometry procedure. This allowed quantitative profiling of neutral and acidic sugar monomers in a complex microbial matrix. Vegetative cells, grown under conditions of phosphate deprivation, contained large amounts of both glucuronic acid and galactosamine (teichuronic acid markers) but not quinovose (a spore component). The results prove that there are three distinct polysaccharides in B. subtilis produced under different physiological growth conditions. PCR amplification was used to examine the 16S/23S rRNA spacer region of each species. The study of this variable genomic region allowed B. subtilis to be differentiated from the B. cereus group of organisms. However, the three B. cereus species produced PCR products that could not be distinguished by low resolution gel electrophoresis. Electrospray mass spectrometry was investigated as a means to obtain precise molecular weight measurements of PCR products. Single base substitutions, additions or deletions could be recognized. It proved vital to remove primers, deoxynucleotides, DNA polymerase and metal ions from the PCR reaction mixture. These reaction components reduced sensitivity and metal ion adduction adversely affected the accuracy of mass measurements. PCR products of 89 base pairs from the B. cereus group were readily differentiated from 114 base pairs products of B. subtilis.