Dental calculus, enamel plaque that has undergone mineralization and adheres to tooth surfaces, can entrap biomolecules and micro-debris that were present prior to mineralization. As such, it serves as a potential reservoir for direct evidence of dietary components. Held in the mineral matrix biomolecules such as DNA and proteins are protected from diagenesis and can be solubilized and analyzed by modern analytical methods. Here, we present a proteomic analysis of calculus samples from the Sanchez Adobe individuals, and compare these results to a small set of 11 Indigenous individuals that pre-date the missions. Bioinformatic analysis of mass spectrometry data identifies elements of both pre- and post-contact diets. Our results show evidence both of the incorporation of introduced foodstuffs, as well as persistence of pre-contact dietary traditions.

Wildlife trafficking is a global issue with devastating ecological effects and synergistic relationships with other forms of international illicit networks. Furs and pelts are a major component of this trade and are more practical to transport. Proper identification of such items is important to be able to prosecute and punish poachers. Wildlife forensic investigators depend on reliable tools and methods to either include or exclude illegal versus legal species. However, due to the chemically and physically harsh production process, DNA-based methods often fail because the sample DNA is degraded. Morphological-based methods have poor species resolution, are time-consuming, and are dependent on a limited number of highly trained individuals. However, these challenges may be by-passed by focusing on the phylogenetic information in protein. DNA changes between closely related species are reflected at the protein level, allowing the phylogenetic information between species to lead to a species-level identification. Proteins are chemically more stable than DNA. Therefore, proteomic analysis of heavily processed items may provide an alternative, and robust, method of species identification. However, determining the amino acid sequences in mass spectrometry data requires the precise sequences to be present in a reference protein database. The hypothesis is that the protein sequences, or reference proteome, of a given species would match more than proteomes from other species and be more efficient at identifying peptide sequences in fur digests from the same species. This research focuses on the phylogenetic relationship between six cat species, lion (Panthera leo), leopard (Panthera pardus), tiger (Panthera tigris), cheetah (Acinonyx jubatus), Canada lynx (Lynx canadensis), and domestic cat (Felis catus); of which the first four had good-quality, morphologically-identified materials that were selected for analysis. The remaining two species, Canada lynx and domestic cat, were included in the phylogenetic comparison using their reference proteome databases. The selected fur or pelt reference material items were digested based on a previously-published method for protein extraction from human hair and analyzed by LC-MS/MS and PEAKS DB software. Species identification was achieved at three different levels: total peptide yield, protein coverage patterns, and presence of peptides containing species-specific markers. For each item sampled, the maximum number of total peptides identified using the PEAKS scoring algorithm was obtained using the corresponding theoretical database. For example, the raw peptides generated from the lion paw pelt yielded 13928 total identified peptides using the lion database. As evolutionary distance between the lion and related felid species increases, the total peptide yield decreases. Using the leopard, tiger, Canada lynx, domestic cat, and cheetah databases, the total peptide yields were less - 13766, 13758, 12604, 12307, and 11774, respectively. Similar results were obtained with the leopard pelt, tiger pelt, and cheetah coat sampled, demonstrating how species identification can be achieved through comparison of total peptide yields. Protein coverage patterns also indicated species of origin. Many keratin and keratin-associated proteins were identified from the hair and skin samples, including, but not limited to, Keratin 14, Keratin 16, Keratin 32, Keratin 35, Keratin 38, Keratin 75, Keratin 82, Keratin 85, Desmoplakin, and Corneodesmosin. Coverage patterns demonstrated the highest percent coverage when analyzing the raw peptides from a sample with its corresponding database. For example, the raw peptides generated from the lion sample that matched with Keratin 35 resulted in 84% coverage using the lion database, while the tiger, leopard, domestic cat, Canada lynx, and cheetah databases resulted in 77%, 75%, 77%, 75%, and 65%, respectively. Species-specific marker locations were identified by protein alignments between the sequences of all six species. Peptides containing these species-specific markers were then identified in the raw data and found to be present only in the samples that correlated with the species of origin. This paper demonstrates four individual examples of species-specific markers within Keratin 35 corresponding to the respective species of origin for all four sampled items: lion, leopard, tiger, and cheetah. In summary, the three approaches used in this project – the total peptide approach, the protein coverage pattern approach, and the species-specific peptide approach – individually demonstrated that species identification is plausible using phylogenetic proteomics. This data demonstrates the robustness and reliability of proteomic approaches to species identification from fur and lays the foundation for future research, such as the development of targeted proteomic assays, for broader application to wildlife forensic casework.

Sexual assault evidence kits (SAEKs) contain physical and biological evidence that is amajor focus of testing in forensic laboratories. There is an intrinsic challenge in processing of SAEK since limited evidence is typically reserved for DNA isolation and human identification. This typically precludes analysis of important contextual information, such as identification of source-material, that requires additional consumption of probative samples. The current best method to process the sexual assault evidence (SAE) starts with a differential extraction where sperm cells are separated from non-sperm cells. The separated fraction goes through the forensic DNA workflow starting with extraction and ending, hopefully, with the perpetrator’s DNA profile. Prior to sample processing, proteins are also contained within biological evidence and can play a major role in body fluid identification. This study focused on the stability and robustness of both DNA and protein signals in SAE across a 5 day time frame. Accordingly, a 1:1 mixture of vaginal fluid and semen was incubated at physiological temperature for 120 hours, as close as an in vitro experiment could reasonably come to actual in vivo evidence. Based on the results obtained, protein and DNA from the mock SAE samples were detectable over the total period. More than half of the original protein, however, was no longer detected after 24 hours, with a 71% decrease (p=0.01) in protein after the full 5-day incubation period. Prostate specific antigen’s (PSA) proteolytic activity did not eliminate large protein structures, such as semenogelin I, throughout the entirety of the incubation. From 0 hours to 120 hours, there was an 87% decrease (p=0.05) in total human DNA and a 78% decrease (p=0.04) in male DNA. Female DNA was no longer detected after 24 hours, but preservation of male DNA was present. We also investigated preservation of proteomic signal during the initial incubation of a differential extraction. There was no change in protein structures and composition in the sample when incubating with dithioerythritol (DTE). When proteinase K was added and incubated with the sample, proteins were degraded in semen, but low molecular weight peptides were still present. These findings infer that proteomic data are still present after proteinase K digestion and that even highly degraded samples may still contain ample source-level information for investigators.

Over the years, government bodies have drafted and enforced numerous species protection acts to combat the issue of illegal wildlife trade. Nonetheless, protected species are still being poached, processed, and trafficked in illicit networks. To support law enforcement agencies, wildlife forensic practitioners aim to identify the species of origin for seized evidentiary items, including feathers. Current methods used in feather identification include morphological and/or DNA-based techniques. While useful, these methodologies have limitations. Feathers lacking distinctive morphological characteristics are difficult to resolve to the species level. Additionally, DNA in feather samples is often compromised or ambiguous as to whether genetic analysis yields any species-specific barcoding information. One way to overcome these limiting issues is by utilizing mass spectrometry (MS)-based proteomics to analyze peptide sequences. One category of abundant proteins that could be used in the proteomic analysis of feathers is keratin, an attractive substrate for analysis and species-identification. This protein class is ubiquitous and does not suffer from sample limitations. In this research, we demonstrate that a digestion protocol optimized for hair can successfully be applied to feathers, and peptides can be retrieved from every feather structure or site (the quill, rachis, vanes, tip, and plumulaceous portion). Additionally, proteomic analysis of the plumulaceous portion of the feather demonstrates that even the site with the lowest average peptide yield can successfully identify five taxonomically informative peptides for the Bald Eagle (Haliaeetus leucocephalus) and 12 taxonomically informative peptides for the Golden Eagle (Aquila chrysaetos; 9/12 being species-specific), with peptides coming from both keratin and non-keratin associated proteins. These species-specific peptides can serve as a basis for methods that identify and resolve species in a forensic context.

Archeological and paleontological bone samples can potentially provide valuable information that can be forensically useful to future research and application. Proteins are robust and do not quickly degrade due to environmental conditions or outside factors. Therefore, understanding potential proteomic signatures in bone is useful when DNA may be too degraded to provide information about the subject. An analysis encompassing bone samples and gelatinous materials was conducted to determine similarities between the two matrices. Radiocarbon dates and ontogenetic ages also compared the samples to identify patterns between the samples. The samples underwent a reduction, alkylation, and trypsin digestion prior to instrumental analysis. The samples were analyzed with an Exploris 480 orbitrap mass spectrometer with an in-line Dionex Ultimate 3K UPLC. The most abundant proteins found in the samples consist of albumin (ALB) and collagen type I (COL1A1). A range of 52-749 peptides (mean 262) and 48-98 proteins (mean 49) were identified in bone samples. The gelatinous material samples identified more peptides with a range of 112 to 754 and an average of 415 peptides. The proteins identified in gelatinous materials range from 13 to 107 groups, averaging 57 proteins. The proteins were categorized into four different groups: blood/serum proteins, extracellular proteins, collagen proteins, and “other” proteins. The bone samples’ proteins were compared based on their ontogenetic age and radiocarbon date. The bone samples were categorized into four groups: Perinate/neonate, 15-23 years old, 24-40 years old, and 40+ in which 47, 37, 15, and 11 exclusive proteins were identified, respectively. The unique and exclusive identified proteins decreased as the ontogenetic age increased. The bone samples were organized into three groups based on their radiocarbon date: 500-1000, 1001-1100, and 1101-1600 years old. The first group (500-1000) identified 97 exclusive proteins. The second group (1001-1100) identified 30 exclusive proteins, and the oldest bones (1101-1600) identified eight exclusive proteins. The gelatinous material samples were compared based on their appearance and structure. The white and feather-like sample contained 31 exclusive proteins, whereas the brown and feather-like identified one protein. The oily and tacky samples identified two exclusive proteins. The paired gelatinous material and bone samples were compared and showed that collagen and albumin prefer the gelatinous material, and most extracellular matrix proteins are found in bone matrix. This paper demonstrates the variability of proteins in bone samples based on their ontogenetic and radiocarbon age. The presence of proteins in gelatinous material with varying physical appearance is also compared to identify potential proteins responsible for physical variability. Overall, the data shows potential gelatinous and bone proteomics use in forensic and bioarcheological spaces.

When people experience food stress or a lack of consistent access to nutritional resources during their life, these stresses often leave osteological markers on the skeleton. During such periods, synthetic processes are compromised and nutrients are often scavenged from other parts of the body, such as bones, to balance competing biological demands. After death, various taphonomic processes will result in decomposition of the body and degradation of the skeletal remains, which eliminate many indicators of starvation and/or markers of physiological stress. However, some of these processes leave osteological signatures that can be detected over archaeological or forensic time frames, particularly in tooth enamel because it is the most robust human tissue. This study, therefore, hypothesizes that the enamel proteome preserves markers of biological stress. Skeletal pathologies noted on ancestral Ohlone skeletal remains from a Late Period (ca. AD 13001700) site in Central California were used to select individuals identified as biologically stressed and compared to individuals with no visible pathologies using Label-Free Quantitative Proteomics. Protein trends correlated with phenotypic expression of biological stress provide more context to the biological processes behind the osteological expressions of those stressors. We demonstrate that biological stress related to starvation resulted in a relative decrease in mineralization proteins, a relative increase in enamel collagen, and changes in the non-specific and targeted immune system. Further, there was no sex-linked bias in biologically stressed individuals that would account for these physiological changes; males and females experienced them equally. This research demonstrates that the enamel proteome preserves a signal of biological stress in archaeological samples, which adds to our anthropological understanding of the lives of people in the past. This method could also be used to identify biological stress in more recent forensic samples.

The use of genetically variant peptides (GVPs) from human hair shaft proteins as a protein-based human identification method has been a validated and discriminating method for the identification of human hair shafts since 2016, with current research showing it can distinguish between identical twins, can produce high random match probabilities (RMP) regardless of hair pigmentation status, age, hair location on the body, and after single harsh chemical treatments. GVPs are a type of single amino acid polymorphism (SAP) found in proteins. These SAPs are classified as GVPs if they are genetically inherited, which can be associated with genetically inherited single nucleotide polymorphisms (SNP) present in at least one percent of the global population. Using GVPs, scientists can infer genotypes, estimate random match probability, and characterize the origin population. This is a complementary human identification method to nuDNA and mtDNA typing. This study exposed human hair shafts from three individuals to oxidative chemistries, specifically to 12 repetitive treatments of Clairol Professional BW 2 powder lightener mixed with Salon Care Professional 50 volume crème developer, and followed by Clairol Professional Beautiful Collection “moisturizing semi-permanent color” in the shade BO1N “Light Natural Blonde.” The protein modifications of interest included cysteic acid, deamidation, and oxidation and di-oxidation of methionine, which are all involved in characterizing oxidative damage to hair. The protein modifications behaved as expected across all subjects and like previous research on oxidative damage and degradation of human hair shafts. The number of GVPs initially increased due to the opening of the matrix from cysteic acid formation and peaked at 4 treatments for all 3 samples. GVPs then decreased as the number of treatments increased to 8 and 12. This was consistent with past oxidative chemistry research on single hairs. The unique GVPs ranged from 4 in the U1.003 control to a maximum of 84 in the U1.016 4-treatment sample. This research supports the findings in previous GVP studies and provides additional validation that protein-based human identification methods can be useful in a forensic context.

DNA analysis of touch evidence on cartridge cases has proven difficult despite numerous attempts to improve collection and extraction methods for this important type of evidence. Numerous factors influence the variability and often paucity of touch evidence such as shedder status, pressure and duration of contact, surface type, and even collection and storage. In addition to these factors, retrieval of biological information from cartridge cases is even more challenging since firing abrades the surface of the case, removing material, and metal in the case can react with DNA, resulting in oxidation and cleavage of the DNA backbone. Protein is also present in biological touch evidence. Protein is more robust chemically than DNA, and it also contains genetic and contextual information that could be useful to investigators. Extraction and analysis of both DNA and proteins from touch evidence can provide the maximum amount of information needed for probative results, especially when DNA is limited. To fully exploit all available biological information on a cartridge case, all types of information need to be transferred from a surface into a technical workflow. Additional methods need to be developed to maximize retrieval of material and to do so in a compatible manner. This study evaluated the efficiency of transferring artificial DNA and protein from a glass microscope slide surface into the workflow for seven different collection and transfer methods: the standard wet-dry cotton swab was compared with alternative methods, a ‘Copan microFLOQ® direct’ swab, with and without lysing agent, and with and without extraction buffer, a cell scraper, and adhesive silicone gel-film sheets. Based on the results obtained, it was determined the ‘Copan microFLOQ® direct’ swab was the overall most efficient and consistent with an average transfer of 58 ± 22% efficiency for DNA and 55 ± 32% for protein compared to 27 ± 24% and 2 ± 27% when using cotton swabs respectively. The evaluation was taken a step further to measure transfer of DNA and protein from real fingermarks on a glass microscope slide to the workflow. It was determined that the cotton swab and the ‘Copan microFLOQ® direct’ swab collected an average of 11.8 ± 20 ng and 15.7 ± 18 ng of total DNA, respectively, and 1200 ± 1200 relative densitometry units (RDU) and 2300 ± 1600 RDU of total relative protein density, respectively. This level of relative variation is high but consistent with previous studies for fingermark deposition. When normalized for individuals the ‘Copan microFLOQ® direct’ increased performance by 1.7-fold (p=0.23) for DNA and 1.9-fold (p=0.02) for protein. Finally, real-life scenarios were mimicked where the cotton swab and ‘Copan microFLOQ® direct’ were used to transfer DNA and protein from unfired and fired cartridge cases into the technical workflow. The cotton swab and the ‘Copan microFLOQ® direct’ swab collected an average of 1.1 ± 1.5 ng and 2.8 ± 2.4 ng of total DNA, respectively, and 660 ± 380 RDU and 1200 ± 1500 RDU of total relative protein density, respectively for unfired cases. When fired there was a significant reduction in the amount of DNA material, but the same trend was observed. The cotton swab and the ‘Copan microFLOQ® direct’ swab collected an average of 0.3 ± 0.5 ng and 0.5 ± 0.6 ng of total DNA, respectively, and 400 ± 340 RDU and 530 ± 450 RDU of total relative protein density. When normalized for individuals the ‘Copan microFLOQ® direct’ increased yields by 3.2-fold (p=0.03) for DNA and 2.0-fold (p=0.06) for protein when unfired and 3.7-fold (p=0.09) for DNA and 1.6-fold (p=0.24) for protein when fired. The firing process was harsher on DNA than on protein. Protein was 2.2-fold (p = 0.07) more persistent than the DNA on cartridge cases after firing. We conclude that transfer of residual biological material from fingermarks is improved when using ‘Copan microFLOQ® direct’ swabs compared to the standard wet-dry cotton swab method, and that the improvement applies to both idealized surfaces such as microscope slides, and challenging surfaces such as unfired and fired cartridge cases. We also observe that protein is more stable during the firing process and potentially can provide additional identifying and contextual information for investigators for this difficult type of evidence.