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.

Proteomic analysis of hair shafts from monozygotic twins: Expression profiles and genetically variant peptides.

Forensic association of hair shaft evidence with individuals is currently assessed by comparing mitochondrial DNA haplotypes of reference and casework samples, primarily for exclusionary purposes. Present work tests and validates more recent proteomic approaches to extract quantitative transcriptional and genetic information from hair samples of monozygotic twin pairs, which would be predicted to partition away from unrelated individuals if the datasets contain identifying information. Protein expression profiles and polymorphic, genetically variant hair peptides were generated from ten pairs of monozygotic twins. Profiling using the protein tryptic digests revealed that samples from identical twins had typically an order of magnitude fewer protein expression differences than unrelated individuals. The data did not indicate that the degree of difference within twin pairs increased with age. In parallel, data from the digests were used to detect genetically variant peptides that result from common nonsynonymous single nucleotide polymorphisms in genes expressed in the hair follicle. Compilation of the variants permitted sorting of the samples by hierarchical clustering, permitting accurate matching of twin pairs. The results demonstrate that genetic differences are detectable by proteomic methods and provide a framework for developing quantitative statistical estimates of personal identification that increase the value of hair shaft evidence.

Structural and biochemical analysis of the Hordeum vulgare L. HvGR-RBP1 protein, a glycine-rich RNA-binding protein involved in the regulation of barley plant development and stress response.

The timing of whole-plant senescence influences important agricultural traits such as yield and grain protein content. Post-transcriptional regulation by plant RNA-binding proteins is essential for proper control of gene expression, development, and stress responses. Here, we report the three-dimensional solution NMR structure and nucleic acid-binding properties of the barley glycine-rich RNA-binding protein HvGR-RBP1, whose transcript has been identified as being >45-fold up-regulated in early-as compared to late-senescing near-isogenic barley germplasm. NMR analysis reveals that HvGR-RBP1 is a multidomain protein comprising a well-folded N-terminal RNA Recognition Motif (RRM) and a structurally disordered C-terminal glycine-rich domain. Chemical shift differences observed in 2D (1)H-(15)N correlation (HSQC) NMR spectra of full-length HvGR-RBP1 and N-HvGR-RBP1 (RRM domain only) suggest that the two domains can interact both in-trans and intramolecularly, similar to what is observed in the tobacco NtGR-RBP1 protein. Further, we show that the RRM domain of HvGR-RBP1 binds single-stranded DNA nucleotide fragments containing the consensus nucleotide sequence 5'-TTCTGX-3' with low micromolar affinity in vitro. We also demonstrate that the C-terminal glycine-rich (HvGR) domain of Hv-GR-RBP1 can interact nonspecifically with ssRNA in vitro. Structural similarities with other plant glycine-rich RNA-binding proteins suggest that HvGR-RBP1 may be multifunctional. Based on gene expression analysis following cold stress in barley and E. coli growth studies following cold shock treatment, we conclude that HvGR-RBP1 functions in a manner similar to cold-shock proteins and harbors RNA chaperone activity. HvGR-RBP1 is therefore not only involved in the regulation of barley development including senescence, but also functions in plant responses to environmental stress.

Structural modification of fentanyls for their retrospective identification by gas chromatographic analysis using chloroformate chemistry.

The one-step breakdown and derivatization of a panel of nine fentanyls to yield uniquely tagged products that can be detected by Electron Ionization Gas Chromatography-Mass Spectrometry (EI-GC-MS) is presented. The method involves the treatment of the synthetic opioids with 2,2,2-trichloroethoxycarbonyl chloride (TrocCl) at 60 °C for 3 h in dichloromethane and furnishes two products from one fentanyl molecule that can be used to retrospectively identify the original opioid. Parameters that were studied and fully optimized for the method included temperature, solvent, nature of scavenging base and reaction time. One of the two resulting products from the reaction bears the trichloroethoxycarbonyl (Troc) tag attached to the norfentanyl portion of the original opioid and greatly aids in the opioid detection and identification process. The methodology has been applied to the chemical modification of a panel of nine fentanyls and in all cases the molecular ion peak for the Troc-norfentanyl product bearing the distinctive trichloroethyl isotopic signature can be clearly observed. The method's LLOD was determined to be 10 ng/mL while its LLOQ was found to be 20 ng/mL. This methodology represents the first application of chloroformates in the chemical modification of this class of synthetic opioids that are notoriously inert to common derivatization strategies available for GC-MS analysis.

Assessing Single-Source Reproducibility of Human Head Hair Peptide Profiling from Different Regions of the Scalp.

Neither microscopical hair comparisons nor mitochondrial DNA sequencing alone, or together, constitutes a basis for personal identification. Due to these limitations, a complementary technique to compare questioned and known hair shafts was investigated. Recently, scientists from Lawrence Livermore National Laboratory's Forensic Science Center and other collaborators developed a peptide profiling technique, which can infer non-synonymous single nucleotide polymorphisms (SNPs) preserved in hair shaft proteins as single amino acid polymorphisms (SAPs). In this study, peptide profiling was evaluated to determine if it can meet forensic expectations when samples are in limited quantities with the possibility that hair samples collected from different areas of a single donor's scalp (i.e., single source) might not exhibit the same SAP profile. The average dissimilarity, percent differences in SAP profiles within each source, ranged from 0% difference to 29%. This pilot study suggests that more work is needed before peptide profiling of hair can be considered for forensic comparisons.

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.

Combined DNA Typing and Protein Identification from Unfired Brass Cartridges,,.

Biological evidence analysis from contact traces is adversely affected by low quantity and quality of DNA. Proteins in these samples contain potentially individualizing information and may be particularly important for difficult surfaces such as brass, where DNA may yield incomplete profiles. In this study, touched unfired brass cartridges were sampled using dry tape or wet swabs and analyzed by separating DNA and protein from the same collected material, thus producing both genomic and proteomic information. DNA recovery was similar for both collection methods, with tape yielding an average of 1.36 ± 1.87 ng and swabs, 1.34 ± 3.04 ng. Analysis by mass spectrometry identified 95 proteins, with the two collection methods showing no significant difference (p = 0.76) in the average number of collected proteins: 44.5 ± 10.9, (tape) versus 47.9 ± 20.4 (swabs). Proteins can be collected from fingerprints at levels necessary to provide identifying information, thus expanding information obtained from challenging evidence.

Proteomic comparison of near-isogenic barley (Hordeum vulgare L.) germplasm differing in the allelic state of a major senescence QTL identifies numerous proteins involved in plant pathogen defense.

Senescence is the last developmental phase of plant tissues, organs and, in the case of monocarpic senescence, entire plants. In monocarpic crops such as barley, it leads to massive remobilization of nitrogen and other nutrients to developing seeds. To further investigate this process, a proteomic comparison of flag leaves of near-isogenic late- and early-senescing barley germplasm was performed. Protein samples at 14 and 21 days past anthesis were analyzed using both two-dimensional gel-based and label-free quantitative mass spectrometry-based ('shotgun') proteomic techniques. This approach identified >9000 barley proteins, and one-third of them were quantified. Analysis focused on proteins that were significantly (p < 0.05; difference ≥1.5-fold) upregulated in early-senescing line '10_11' as compared to late-senescing variety 'Karl', as these may be functionally important for senescence. Proteins in this group included family 1 pathogenesis-related proteins, intracellular and membrane receptors or co-receptors (NBS-LRRs, LRR-RLKs), enzymes involved in attacking pathogen cell walls (glucanases), enzymes with possible roles in cuticle modification, and enzymes involved in DNA repair. Additionally, proteases and elements of the ubiquitin-proteasome system were upregulated in line '10_11', suggesting involvement of nitrogen remobilization and regulatory processes. Overall, the proteomic data highlight a correlation between early senescence and upregulated defense functions. This correlation emerges more clearly from the current proteomic data than from a previously performed transcriptomic comparison of 'Karl' and '10_11'. Our findings stress the value of studying biological systems at both the transcript and protein levels, and point to the importance of pathogen defense functions during developmental leaf senescence.

¹H, ¹³C, ¹5N backbone and side chain NMR resonance assignments for the N-terminal RNA recognition motif of the HvGR-RBP1 protein involved in the regulation of barley (Hordeum vulgare L.) senescence.

Leaf senescence is an important process in the developmental life of all plant species. Senescence efficiency influences important agricultural traits such as grain protein content and plant growth, which are often limited by nitrogen use. Little is known about the molecular mechanisms regulating this highly orchestrated process. To enhance our understanding of leaf senescence and its regulation, we have undertaken the structural and functional characterization of previously unknown proteins that are involved in the control of senescence in barley (Hordeum vulgare L.). Previous microarray analysis highlighted several barley genes whose transcripts are differentially expressed during senescence, including a specific gene which is greater than 40-fold up-regulated in the flag leaves of early- as compared to late-senescing near-isogenic barley lines at 14 and 21 days past flowering (anthesis). From inspection of its amino acid sequence, this gene is predicted to encode a glycine-rich RNA-binding protein herein referred to as HvGR-RBP1. HvGR-RBP1 has been expressed as a recombinant protein in Escherichia coli, and preliminary NMR data analysis has revealed that its glycine-rich C-terminal region [residues: 93-162] is structurally disordered whereas its N-terminal region [residues: 1-92] forms a well-folded domain. Herein, we report the complete (1)H, (13)C, and (15)N resonance assignments of backbone and sidechain atoms, and the secondary structural topology of the N-terminal RNA recognition motif (RRM) domain of HvGR-RBP1, as a first step to unraveling its structural and functional role in the regulation of barley leaf senescence.

Attenuation of ephrinB2 reverse signaling decreases vascularized area and preretinal vascular tuft formation in the murine model of oxygen-induced retinopathy.

PURPOSE: EphB4 and ephrinB2 are known key regulators of retinal vascular development, but due to their capacity for bidirectional signaling, delineation of their individual roles in this process remains unclear. To better dissect out individual contributions, a model of proliferative retinopathy in mice with attenuated ephrinB2 reverse signaling was studied. It was hypothesized that endothelial ephrinB2 reverse signaling regulates hypoxia-induced capillary sprouting, as well as the pathologic formation of neovascular tufts in postnatal retinal microvascular networks. METHODS: Genetically manipulated mice with attenuated ephrinB2 reverse signaling (ephrinB2(lacZ/+)), along with wild-type (WT) controls, were exposed to oxygen-induced retinopathy (OIR), a postnatal model of proliferative retinopathy. At peak disease (postnatal day 18), microvascular networks were analyzed to examine intraretinal revascularization, capillary sprouting, and pathologic neovascularization responses. EphB4 and phosphorylated ephrinB protein expression patterns along retinal microvessels were also assessed. RESULTS: EphrinB2(lacZ/+) mice exhibited reduced hypoxia-induced revascularization (P ≤ 0.04) and reduced formation of neovascular tufts (P < 0.001), as compared with WT controls. Corresponding to the observed inhibition of retinal angiogenesis, ephrinB2(lacZ/+) retinas displayed an increased number of blind-ended capillary sprout tips (P < 0.02) and endothelial filopodial processes (P = 0.001). In WT and ephrinB2(lacZ/+) OIR-exposed retinas, ephrinB was confined to endothelial cells, with expression detected along angiogenic vascular processes including neovascular tufts and blind-ended capillary sprouts. CONCLUSIONS: EphrinB2 reverse signaling is a regulator of key processes during retinal vascularization and controls pathologic retinal angiogenesis through direct effects on capillary sprouting and endothelial filopodia formation.