Proteomic Characterization of Damaged Single Hairs Recovered after an Explosion for Protein-Based Human Identification.

Evidence recovery is challenging where an explosion has occurred. Though hair evidence may be sufficiently robust to be recovered at the site, forensic analysis underutilizes the matrix by relying on morphological analysis. Where DNA is compromised, particularly in hair, protein-based human identification presents a promising alternative. Detection of amino acid polymorphisms in hair proteins as genetically variant peptides (GVPs) permits the inference of individualizing single nucleotide polymorphisms for identification. However, an explosive blast may damage hair proteins and compromise GVP identification. This work assesses effects of an explosive blast on the hair proteome and GVP identification, investigates microscopy as a predictor of proteome profiling success in recovered hairs to improve analysis throughput, and quantifies discriminative power in damaged hairs. The proteomics dataset has been deposited into the ProteomeXchange Consortium (PXD017427). With the exception of degradation in keratins K75, K80, K40, and keratin-associated protein KAP10-11 as markers of hair cuticular damage, corroborated by scanning electron microscopic analysis, minimal hair proteome degradation following explosion allowed successful proteome profiling of single hairs regardless of morphological damage. Finally, GVP identification remained independent of explosion conditions, permitting similar discriminative power between exploded and undamaged hairs. These findings lend greater confidence to GVP analysis in one-inch hairs for forensic identification and provide information about hair protein localization.

Rapid and mild silylation of ß-amino alcohols at room temperature mediated by N-methylimidazole for enhanced detectability by gas chromatography/electron ionization mass spectrometry.

RATIONALE: In this work, we expand the use of in situ activation of chloro(dimethyl)phenylsilane using N-methylimidazole (NMI) for the effective derivatization of ß-aminoethyl alcohols. Due to its enhanced nucleophilic character, NMI is expected to act as an efficient activator in these reactions. METHODS: The derivatization of a panel of ß-aminoethyl alcohols was accomplished by reacting the analyte with chloro(dimethyl)phenylsilane in the presence of either NMI or pyridine. After the addition of chloro(dimethyl)phenylsilane, the vials were gently tumbled for 1 h at ambient temperature. The phenyldimethylsilyl derivatives were identified using gas chromatography/electron ionization mass spectrometry (GC/EI-MS). RESULTS: A total of ten ß-aminoethyl alcohols were successfully derivatized via in situ activation of chloro(dimethyl)-phenylsilane with NMI. Derivatization with NMI was significantly more efficient than with pyridine by a factor of 3-6 for the studied alcohols. The derivatizations in the presence of NMI were found to occur in just 1 h and were conveniently executed at ambient temperature. CONCLUSIONS: The use of the nitrogenous base NMI in order to activate chloro(dimethyl)phenylsilane for the efficient silylation of a panel of ß-aminoethyl alcohols has been demonstrated. The present work shows that NMI is an efficient base for the smooth derivatization of these types of alcohols. Furthermore, the installation of the bulky PDMS group onto these alcohols adds to the certainty that this is a viable approach for the installation of the more commonly employed, trimethylsilyl group. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

31P-edited diffusion-ordered 1H NMR spectroscopy for the spectral isolation and identification of organophosphorus compounds related to chemical weapons agents and their degradation products.

Organophosphorus compounds represent a large class of molecules that include pesticides, flame-retardants, biologically relevant molecules, and chemical weapons agents (CWAs). The detection and identification of organophosphorus molecules, particularly in the cases of pesticides and CWAs, are paramount to the verification of international treaties by various organizations. To that end, novel analytical methodologies that can provide additional support to traditional analyses are important for unambiguous identification of these compounds. We have developed an NMR method that selectively edits for organophosphorus compounds via (31)P-(1)H heteronuclear single quantum correlation (HSQC) and provides an additional chromatographic-like separation based on self-diffusivities of the individual species via (1)H diffusion-ordered spectroscopy (DOSY): (1)H-(31)P HSQC-DOSY. The technique is first validated using the CWA VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate) by traditional two-dimensional DOSY spectra. We then extend this technique to a complex mixture of VX degradation products and identify all the main phosphorus-containing byproducts generated after exposure to a zinc-cyclen organometallic homogeneous catalyst.

Automated analysis of scanning electron microscopic images for assessment of hair surface damage.

Mechanical damage of hair can serve as an indicator of health status and its assessment relies on the measurement of morphological features via microscopic analysis, yet few studies have categorized the extent of damage sustained, and instead have depended on qualitative profiling based on the presence or absence of specific features. We describe the development and application of a novel quantitative measure for scoring hair surface damage in scanning electron microscopic (SEM) images without predefined features, and automation of image analysis for characterization of morphological hair damage after exposure to an explosive blast. Application of an automated normalization procedure for SEM images revealed features indicative of contact with materials in an explosive device and characteristic of heat damage, though many were similar to features from physical and chemical weathering. Assessment of hair damage with tailing factor, a measure of asymmetry in pixel brightness histograms and proxy for surface roughness, yielded 81% classification accuracy to an existing damage classification system, indicating good agreement between the two metrics. Further ability of the tailing factor to score features of hair damage reflecting explosion conditions demonstrates the broad applicability of the metric to assess damage to hairs containing a diverse set of morphological features.

Development of a Protein-based Human Identification Capability from a Single Hair.

Shed human hair (lacking root nuclear DNA) frequently contributes important information to forensic investigations involving human identification. Detection of genetic variation observed in amino acid sequences of hair proteins provides a new suite of identity markers that augment microscopic hair analysis and mitochondrial DNA sequencing. In this study, a new method that completely dissolves single hairs using a combination of heat, ultrasonication, and surfactants was developed. Dissolved proteins were digested and genetically variant peptide (GVP) profiles were obtained for single hairs (25 mm) via high-resolution nanoflow liquid chromatography-based mass spectrometry and a novel exome-driven bioinformatic approach. Overall, 6519 unique peptides were identified and a total of 57 GVPs were confirmed. Random match probabilities ranged between 2.6 × 10-2 and 6.0 × 10-9 . The new bioinformatic strategy and ability to analyze GVPs in forensically relevant samples sizes demonstrate applicability of this approach to distinguish individuals in forensic contexts.

Hair Proteome Variation at Different Body Locations on Genetically Variant Peptide Detection for Protein-Based Human Identification.

Human hair contains minimal intact nuclear DNA for human identification in forensic and archaeological applications. In contrast, proteins offer a pathway to exploit hair evidence for human identification owing to their persistence, abundance, and derivation from DNA. Individualizing single nucleotide polymorphisms (SNPs) are often conserved as single amino acid polymorphisms in genetically variant peptides (GVPs). Detection of GVP markers in the hair proteome via high-resolution tandem mass spectrometry permits inference of SNPs with known statistical probabilities. To adopt this approach for forensic investigations, hair proteomic variation and its effects on GVP identification must first be characterized. This research aimed to assess variation in single-inch head, arm, and pubic hair, and discover body location-invariant GVP markers to distinguish individuals. Comparison of protein profiles revealed greater body location-specific variation in keratin-associated proteins and intracellular proteins, allowing body location differentiation. However, robust GVP markers derive primarily from keratins that do not exhibit body location-specific differential expression, supporting GVP identification independence from hair proteomic variation at the various body locations. Further, pairwise comparisons of GVP profiles with 8 SNPs demonstrated greatest interindividual variation and high intraindividual consistency, enabling similar differentiative potential of individuals using single hairs irrespective of body location origin.

Chemical vapor discrimination using a compact and low-power array of piezoresistive microcantilevers.

A compact and low-power microcantilever-based sensor array has been developed and used to detect various chemical vapor analytes. In contrast to earlier micro-electro-mechanical systems (MEMS) array sensors, this device uses the static deflection of piezoresistive cantilevers due to the swelling of glassy polyolefin coatings during sorption of chemical vapors. To maximize the sensor response to a variety of chemical analytes, the polymers are selected based on their Hildebrand solubility parameters to span a wide range of chemical properties. We utilize a novel microcontact spotting method to reproducibly coat a single side of each cantilever in the array with the polymers. To demonstrate the utility of the sensor array we have reproducibly detected 11 chemical vapors, representing a breadth of chemical properties, in real time and over a wide range of vapor concentrations. We also report the detection of the chemical warfare agents (CWAs) VX and sulfur mustard (HD), representing the first published report of CWA vapor detection by a polymer-based, cantilever sensor array. Comparisons of the theoretical polymer/vapor partition coefficient to the experimental cantilever deflection responses show that, while general trends can be reasonably predicted, a simple linear relationship does not exist.

Static deflection measurements of cantilever arrays reveal polymer film expansion and contraction.

An optical static method of detection is used to interpret surface stress induced bending related to cantilevers coated on one side with poly(vinyl alcohol), poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate), and poly(vinyl chloride-co-vinyl acetate-co-2-hydroxypropyl acrylate), or respectively, PVA, PVB, and PVC, and exposed to various solvent vapors. Results indicate that the adsorption and surface interactions of the different solvent vapors that cause polymer swelling and shrinking lead to rearrangements, which have been shown to change the elastic properties of the polymer film, and subsequently, the spring constant of the polymer coated cantilever. Static deflection measurements allow the direction of cantilever bending to be determined, which adds a new dimension of usefulness for surface functionalized cantilevers as transducers in the development of novel microelectromechanical systems (MEMS).

Demonstration of Protein-Based Human Identification Using the Hair Shaft Proteome.

Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects' DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European-American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.

Hydrolysis of acetylcholinesterase inhibitors--organophosphorus acid anhydrolase enzyme immobilization on photoluminescent porous silicon platforms.

We report on the immobilization of an OPAA enzyme on luminescent porous silicon devices, and on the utilization of this new platform to hydrolyze p-nitrophenyl-soman.

New method for attachment of biomolecules to porous silicon.

Biomolecules have been attached to porous silicon by a new linking method that forms a direct Si-C bond on the surface and retains the photoluminescence of the porous silicon.

Functionalized silicon membranes for selective bio-organism capture.

Membranes with various pore size, length, morphology and density have been synthesized from diverse materials for size-exclusion-based separation. An example is the sterilization of intravenous lines by exclusion of bacteria and viruses using polyvinylidene fluoride membranes with 0.1-microm-diameter pores. Chemically specific filtration has recently been addressed for small molecules. Nevertheless, specific bio-organism immobilization and detection remains a great technical challenge in many biomedical applications, such as decontamination or analysis of air and liquids such as drinking water and body fluids. To achieve this goal, materials with controlled pore diameter, length and surface chemistry are required. In this letter, we present the first functionalized silicon membranes and demonstrate their ability to selectively capture simulated bio-organisms. These extremely versatile and rigid devices open the door to a new class of materials that are able to recognize the external fingerprints of bio-organisms-such as size and outer membrane proteins-for specific capture and detection applications.