Protein Residue Analysis Research Articles

The potential of aptamers for the analysis of ceramic bound proteins found within pottery

Archaeological pottery are the most numerous objects found during excavations and reflect the culinary practices of the past. However, their functionality for cooking/storing specific foods or drinks cannot be deduced solely from comparing their shapes and sizes. Analysis of protein residues bound to ceramics can reveal the protein/animal type through their amino acid sequence, thus enabling direct identification of food types. Therefore, the aim of our experimental study was to test sixteen aptamers for the analysis of proteinaceous organic residues found within the porous structure of pottery. Traditionally prepared archaeological ceramic replicas were cooked for 5 days in various food/protein suspensions, were UV aged, buried for a year, excavated, and extensively cleaned. Their shards were analysed using immunofluorescence microscopy with aptamers. Results show that eight aptamers (Clone1 and Kirby for egg residuals; seqU5 and BLG14 for milk residuals; HA for blood residuals; Gli4 for gluten residuals; Par1 for fish residuals; and D1 for collagen residuals) produced a successful/specific immunofluorescence microscopy result when they were hybridised to shards containing target protein residuals. Interestingly, on whole egg control samples, when the egg lysozyme-targeting aptamer Kirby was used, fluorescence intensity was 3.1 times greater compared to that observed with anti-ovalbumin antibodies.

Technological and Protein Residue Analysis on Ancient Stemmed Projectile Points of the Southern Andes Highlands

ABSTRACT We present the results of technological and protein residue analyses of the lithic assemblages recovered at the Pedernales-38 site (26.5°S; 69°W), located in the highlands of the southern Andes (3380 masl). Based on a reconstruction of the operational chains and the differential representation of their phases, we suggest that the final production, use, maintenance, and discard of large stemmed projectile points occurred at the site. Of the assemblage studied, nine (9) projectile points and one end-scraper were subjected to residue analysis using cross-over immunoelectrophoresis (CIEP). Four positive reactions to antisera of human, camelid (guanaco or vicuña) and canid (Andean fox) were recorded. The results are discussed based on the depositional context and the structure of the archeological record. We propose that Pedernales projectile points were mainly used in hunting weapons to capture wild camelids during the seasonal occupation of the Andes highlands.

Landscape-Based Mutational Sensitivity Cartography and Network Community Analysis of the SARS-CoV-2 Spike Protein Structures: Quantifying Functional Effects of the Circulating D614G Variant.

We developed and applied a computational approach to simulate functional effects of the global circulating mutation D614G of the SARS-CoV-2 spike protein. All-atom molecular dynamics simulations are combined with deep mutational scanning and analysis of the residue interaction networks to investigate conformational landscapes and energetics of the SARS-CoV-2 spike proteins in different functional states of the D614G mutant. The results of conformational dynamics and analysis of collective motions demonstrated that the D614 site plays a key regulatory role in governing functional transitions between open and closed states. Using mutational scanning and sensitivity analysis of protein residues, we identified the stability hotspots in the SARS-CoV-2 spike structures of the mutant trimers. The results suggest that the D614G mutation can induce the increased stability of the open form acting as a driver of conformational changes, which may result in the increased exposure to the host receptor and promote infectivity of the virus. The network community analysis of the SARS-CoV-2 spike proteins showed that the D614G mutation can enhance long-range couplings between domains and strengthen the interdomain interactions in the open form, supporting the reduced shedding mechanism. This study provides the landscape-based perspective and atomistic view of the allosteric interactions and stability hotspots in the SARS-CoV-2 spike proteins, offering a useful insight into the molecular mechanisms underpinning functional effects of the global circulating mutations.

Confirming a Cultural Association at the La Prele Mammoth Site (48CO1401), Converse County, Wyoming

Archaeologists have long subjected Clovis megafauna kill/scavenge sites to the highest level of scrutiny. In 1987, a Columbian mammoth (Mammuthus columbi) was found in spatial association with a small artifact assemblage in Converse County, Wyoming. However, due to the small tool assemblage, limited nature of the excavations, and questions about the security of the association between the artifacts and mammoth remains, the site was never included in summaries of human-killed/scavenged megafauna in North America. Here we present the results of four field seasons of new excavations at the La Prele Mammoth site that confirm the presence of an associated cultural occupation based on geologic context, artifact attributes, spatial distributions, protein residue analysis, and lithic microwear analysis. This new work identified a more extensive cultural occupation including the presence of multiple discrete artifact clusters in close proximity to the mammoth bone bed. This study confirms the presence of a second Clovis mammoth kill/scavenge site in Wyoming and shows the value in revisiting proposed terminal Pleistocene kill/scavenge sites.

Taphonomy and negative results: An integrated approach to ceramic-bound protein residue analysis

Despite the growing body of evidence demonstrating that proteins can survive for thousands to even millions of years in selected contexts, there are relatively few examples of the successful recovery and identification of archaeological protein residues from ceramic artifacts. Claims of positive results are sometimes contentious and frequently challenged. One source of confusion in the debate is a general lack of consideration for the taphonomic histories of ceramic-bound proteins. To gain insight into this issue, we conducted an integrated, mass spectrometry-based study examining ceramic-bound protein that was experimentally aged over the course of 12 months. Results demonstrate the rapid degradation of proteins, raise questions about the degree to which ceramic-bound proteins can be expected to survive over time, and reveal some of the limitations of non-targeted mass spectrometry-based analyses. Further, by comparing results from our experimentally-aged samples to the those we obtained from a multi-pronged study of archaeological ceramics from the American Southwest, we are able to draw more confident conclusions regarding our lack of meaningful matches in the archaeological samples.

FOLSOM MAMMOTH HUNTERS? THE TERMINAL PLEISTOCENE ASSEMBLAGE FROM OWL CAVE (10BV30), WASDEN SITE, IDAHO

The 1960s and 1970s excavations at Owl Cave (10BV30) recovered mammoth bone and Folsom-like points from the same strata, suggesting evidence for a post-Clovis mammoth kill. However, a synthesis of the excavation data was never published, and the locality has since been purged from the roster of sites with human/extinct megafauna associations. Here, we present dates on bone from the oldest stratum, review provenience data, conduct a bone-surface modification study, and present the results of a protein-residue analysis. Our study fails to make the case for mammoth hunting by Folsom peoples. Although two of the point fragments tested positive for horse or elephant protein, recent AMS dates indicate that all of the mammoth remains predate Folsom, and horse remains are absent from the Owl Cave collection. Further, no unambiguously cultural surface modifications were identified on any of the mammoth remains. Given the available data, the Owl Cave deposits are most parsimoniously read as containing a Folsom-age occupation in a buried context, the first of its kind in the desert West, but one nonetheless part of a palimpsest of terminal Pleistocene materials.

Confronting complexities of artifact–geofact debates: re-analysis of a coarse volcanic rock assemblage from Chilean Patagonia

An assemblage of coarse volcanic rock (CVR) derived from the walls of a rockshelter in Aisén, Chile was recently described as consisting of bifaces, projectile points, and picks [Prentiss et al. 2015a. Lithic Technology 40:112–127]. The objects are not universally accepted as cultural, however, on account of both their crude appearance and the fact that CVR industries have not been previously identified in either the valley where the objects were found or elsewhere in Patagonia. We present the results of three independent studies—a single-blind poll of lithics specialists, on-site assessment of stone availability and natural agents of modification, and protein residue analysis using cross-over immunoelectrophoresis—none of which support a cultural origin for the CVR specimens. We discuss these findings in terms of a more general debate in lithics analysis regarding reconciliation of conflicting evidence and interpretations in ambiguous cases.

CoeViz: a web-based tool for coevolution analysis of protein residues.

BackgroundProteins generally perform their function in a folded state. Residues forming an active site, whether it is a catalytic center or interaction interface, are frequently distant in a protein sequence. Hence, traditional sequence-based prediction methods focusing on a single residue (or a short window of residues) at a time may have difficulties in identifying and clustering the residues constituting a functional site, especially when a protein has multiple functions. Evolutionary information encoded in multiple sequence alignments is known to greatly improve sequence-based predictions. Identification of coevolving residues further advances the protein structure and function annotation by revealing cooperative pairs and higher order groupings of residues.ResultsWe present a new web-based tool (CoeViz) that provides a versatile analysis and visualization of pairwise coevolution of amino acid residues. The tool computes three covariance metrics: mutual information, chi-square statistic, Pearson correlation, and one conservation metric: joint Shannon entropy. Implemented adjustments of covariance scores include phylogeny correction, corrections for sequence dissimilarity and alignment gaps, and the average product correction. Visualization of residue relationships is enhanced by hierarchical cluster trees, heat maps, circular diagrams, and the residue highlighting in protein sequence and 3D structure. Unlike other existing tools, CoeViz is not limited to analyzing conserved domains or protein families and can process long, unstructured and multi-domain proteins thousands of residues long. Two examples are provided to illustrate the use of the tool for identification of residues (1) involved in enzymatic function, (2) forming short linear functional motifs, and (3) constituting a structural domain.ConclusionsCoeViz represents a practical resource for a quick sequence-based protein annotation for molecular biologists, e.g., for identifying putative functional clusters of residues and structural domains. CoeViz also can serve computational biologists as a resource of coevolution matrices, e.g., for developing machine learning-based prediction models. The presented tool is integrated in the POLYVIEW-2D server (http://polyview.cchmc.org/) and available from resulting pages of POLYVIEW-2D.

Early Hunter-Gatherer Tool Use and Animal Exploitation: Protein and Microwear Evidence from the Central Savannah River Valley

AbstractResults of protein residue and lithic microwear analyses are reported for Paleoindian and Early Archaic stone tools from a Carolina bay sand rim on the Aiken Plateau of South Carolina, USA. Protein residue analysis is performed using crossover Immunoelectrophoresis (CIEP), and indicates positive results for Bovidae, Cervidae, Galliformes, and Meleagris gallopavo. These results are complemented by a larger immunological study of 135 diagnostic hafted bifaces from South Carolina and Georgia. Among other species identified, bovid residue was found on multiple Paleoindian hafted bifaces, an Early Archaic hafted biface, and a Middle Archaic hafted biface. Results suggest continuity of species selection and availability across the Pleistocene/Holocene boundary and provide no support for the exploitation of extinct fauna. The data do provide compelling evidence for a demographic shift and/or regional extirpation of Bovidae possibly as late as the early mid-Holocene in the Southeast. In addition, microwear analysis of artifacts from Flamingo Bay indicate intensive hide scraping, antler boring, bone graving/planing/pointing, wood whittling, and hafting traces. Microwear data suggest intentional snap-fracture or bipolarization of exhausted or broken Clovis points for reuse as hide scrapers, and use of large bifacial knives and unifacial scrapers in intensive defleshing activities consistent with large animal butchery.

Validation of a Non-Targeted LC-MS Approach for Identifying Ancient Proteins: Method Development on Bone to Improve Artifact Residue Analysis

Identification of protein residues from prehistoric cooking pottery using mass spectrometry is challenging because proteins are removed from original tissues, are degraded from cooking, may be poorly preserved due to diagenesis, and occur in a palimpsest of exogenous soil proteins. In contrast, bone proteins are abundant and well preserved. This research is part of a larger method-development project for innovation and improvement of liquid chromatography – mass spectrometry analysis of protein residues from cooking pottery; here we validate the potential of our extraction and characterization approach via application to ancient bone proteins. Because of its preservation potential for proteins and given that our approach is destructive, ancient bone identified via skeletal morphology represents an appropriate verification target. Proteins were identified from zooarchaeological turkey (Meleagris gallopavo Linnaeus Phasianidae), rabbit (Lagomorpha), and squirrel (Sciuridae) remains excavated from ancient pueblo archaeological sites in southwestern Colorado using a non-targeted LC-MS/MS approach. The data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD002440. Improvement of highly sensitive targeted LC-MS/MS approaches is an avenue for future method development related to the study of protein residues from artifacts such as stone tools and pottery.

Haskett Spear Weaponry and Protein-Residue Evidence of Proboscidean Hunting in the Great Salt Lake Desert, Utah

Recent finds in the Great Salt Lake Desert provide new evidence about Haskett projectile-point technology. Haskett is a large, early representative of the Western Stemmed Tradition. The collection includes the longest Haskett specimen yet documented and another that has yielded a positive result to elephant antiserum via protein-residue analysis. Technological evidence is provided that supports the interpretation of Haskett points as sophisticated throwing/thrusting spear tips.

Serine protease identification (in vitro) and molecular structure predictions (in silico) from a phytopathogenic fungus,Alternaria solani

Serine proteases are involved in an enormous number of biological processes. The present study aims at characterizing three-dimensional (3D) molecular architecture of serine proteases from early blight pathogen, Alternaria solani that are hypothesized to be markers of phytopathogenicity. A serine protease was purified to homogeneity and MALDI-TOF-MS/MS analysis revealed that protease produced by A. solani belongs to alkaline serine proteases (AsP). AsP is made up of 403 amino acid residues with molecular weight of 42.1 kDa (Isoelectric point - 6.51) and its molecular formula was C1859 H2930 N516 O595 S4 . AsP structure model was built based on its comparative homology with serine protease using the program, MODELER. AsP had 16 β-sheets and 10 α-helices, with Ser(350) (G347-G357), Asp(158) (D158-H169), and His(193) (H193-G203) in separate turn/coil structures. Biological metal binding region situated near 6th-helix and His(193) residue is responsible for metal binding site. Also, calcium ion (Ca(2+)) is coordinated by the carboxyl groups of Lys(84), Ile(85), Lys(86), Asp(87), Phe(88), Ala(89), Ala(90) (K84-A90) for first Ca(2+) binding site and carbonyl oxygen atom of Lys(244), Gly(245), Arg(246), Thr(247), Lys(248), Lys(249), and Ala(250) (K244-A250), for second Ca(2+) binding site. Moreover, Ramachandran plot analysis of protein residues falling into most favored secondary structures were determined (83.3%). The predicted molecular 3D structural model was further verified using PROCHECK, ERRAT, and VADAR servers to confirm the geometry and stereo-chemical parameters of the molecular structural design. The functional analysis of AsP 3D molecular structure predictions familiar in the current study may provide a new perspective in the understanding and identification of antifungal protease inhibitor designing.

Late Pleistocene protein residues from the Mahaffy cache, Colorado

This paper reports the analysis of protein residues from tools recovered in a cache within the city limits of Boulder, Colorado, USA. This cache included a total of 83 artifacts, all of which we subjected to cross-over immunoelectrophoresis (CIEP). Four of the 83 produced results, with residues from each of these reacting with antigens from a different taxon: one tool shows evidence of use on sheep, one on bear, one on horse, and one on camel. Varieties of sheep and bear have been present in Colorado throughout human history, but horses and camelids have been in the state either during the Pleistocene or the last 200 years. Several lines of evidence indicate that the cache cannot be recent, and our CIEP results therefore imply that the cache date to the late Pleistocene. Typological aspects of the artifacts in the cache are consistent with artifacts known to be Clovis, and the combination of CIEP and typological data thus indicate that the cache is Clovis as well. These data contribute to an increasing dataset documenting the broad range of animals other than elephants hunted by Clovis groups in North America.

An Optimized Approach for Protein Residue Extraction and Identification from Ceramics After Cooking

Extraction of protein residues from archaeological matrices, such as pottery clay, lithics, and grinding stones, has proven to be methodologically challenging. Protein residue analysis is fraught with technical challenges in analytical chemistry. In cooking pottery, protein residues are thought to bind to clay surfaces in vessel walls through a variety of primarily non-covalent interactions. Removal of protein residues requires the disruption of these interactions, and a diverse set of tools has been proposed and applied. Here, we test extraction procedures through varying combinations of physical parameters and solvents to derive an optimal approach yielding efficiencies of recovery from experimental pottery above 60%. The utility of our extraction approach was further validated through liquid chromatography–mass spectrometry analysis of experimental residues. We have identified several hurdles to developing a successful study of protein residues from pottery, each of which is surmountable with additional method development in the realm of archaeological chemistry.

Dealing with the identification of protein species in ancient amphorae

This manuscript deals with the identification of protein residues in amphorae, including particularly identification of protein species. The work described was performed on fishes, the anchovy (Engraulis encrasicolus) and bonito (Sarda sarda) species frequently found in the Mediterranean area. Based on proteomic techniques, the analytical strategy was adapted to analysis of protein residues from tiny ceramic fragments. The major difficulty was to extract proteins and limit their hydrolysis during the sample preparation; consequently, multiple soft extraction techniques were evaluated. The most valuable results were obtained using a solution containing high amounts of denaturing agents, urea and thiourea, reducing agent, dithiothreitol, and detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The analysis using nano liquid chromatography-nano electrospray ionization double quadrupole time-of-flight mass spectrometry resulted in the identification of up to 200 proteins for the anchovy and bonito species, among which 73 peptides were found to be fish-specific. Because bonito and anchovy species are not documented and fully sequenced in genomic databases, the preliminary protein identification was realized via sequence homology to other fish sequenced species. Amino acid substitutions of peptides were assigned on the basis of the interpretation of tandem mass spectrometry spectra using de novo sequencing; these peptides, not reported up to now in databases, constitute species-specific markers. The method developed was finally applied to an archaeological sample replica impregnated with a mixture of fish tissue from both species; this experiment successfully led to the identification of 17 fish proteins, including 33 fish-specific peptides. This work shows that the analytical method developed has great potential for the identification of protein species in complex archaeological samples.

Development of a validated process for manual preparation of dental transmission instruments

The goal of the study was to develop a validated manual preparation process that conforms to the requirements of validation guidelines. Twelve dental transmission devices from various manufacturers (turbines, handpieces, and contra-angle handpieces) were artificially contaminated with bovine hemoglobin for the test. Ten microliters (corresponding to 800 μg) of bovine hemoglobin solution (concentration 80 mg/ml) was pipetted into the spray water and spray air channels. The manual preparation was conducted by blowing air through the spray channels of the transmission instruments through an attachment to a treatment unit (model 1060T, KaVo, Biberach, Germany) for 5 s. The spray channels were cleaned with WL-Clean (Alpro, Georgen, Germany) as directed by the manufacturer. The spray channels were disinfected with WL-Cid (Alpro) and the spray channels were blow-dried with WL-Dry (Alpro) at the end of the exposure time as directed by the manufacturer. To determine the protein content (protein residue analysis) in the channels of the transmission instruments, 2 ml of an alkaline SDS solution (1%; pH 11) was flushed through the channels. For the quantitative protein residue analysis, the Biuret method was used as described in DIN EN 15883-1:2006. After the application of this method, all results of the protein residue analysis were within the acceptance criteria of the validation guideline. The newly developed manual preparation process is therefore confirmed as suitable from a hygienic viewpoint for preparation of transmission instruments in the dental practice.

Integration of use-wear with protein residue analysis – a study of tool use and function in the south Scandinavian Early Neolithic

This study presents research on how an integration of use-wear analysis with protein residue analysis can produce new results on prehistoric tool use and function. Thirty flint artefacts from an Early Neolithic TRB site in south Sweden were analysed for both use-wear and protein residues. The results show a positive correlation between the two methods. The Early Neolithic tools analysed were primarily used to process fish.

Analysis Of Influenza Hemagglutinin Ligand-binding From Mutational Data And Molecular Motion

Influenza hemagglutinin binds to sialic-acid-terminated glycans on the surface of target cells. This ligand-binding specificity of hemagglutinin is believed an important determinant of which host species it infects. Site-directed mutagenesis of hemagglutinin, expression, and determination of ligand-binding affinity are feasible but technically involved, so only a few hemagglutinin mutations have been tested in this manner. To understand the mechanism of binding specificity and guide further experiments, we have analyzed H5N1 avian influenza isolates to predict residues important to ligand binding and ultimately to ligand specificity. We employed sequence data from all available isolates in combination with analysis of protein residues that correlate with ligand conformation in molecular dynamics simulations to generate candidate sites for mutation. Using this combined analysis, we have predicted five residues both in the sialic-acid-binding site of hemagglutinin and more distant from it. In an initial evaluation, we have performed extensive molecular dynamics simulation of twelve point mutations at these sites. We simulated each of the 12 mutants in 3x100 ns and 200x10 ns simulations to obtain more robust statistical estimates of ligand dissociation. These simulations indicate a greatly increased dissociation rate from the mutants compared to simulations of wild-type H5N1 hemagglutinin VN1194, indicating that the mutations may disrupt ligand binding as expected. This analytic technique may thus provide an important means of screening potential binding-specificity mutants of influenza hemagglutinin as well as a more general tool to assess residues involved in ligand binding.

Effects of microwave irradiation on ruminal protein and starch degradation of corn grain

This study was designed to evaluate effects of 800 W microwave irradiation for 3, 5 and 7 min on ruminal dry matter (DM), crude protein (CP) and starch degradation parameters of corn grain. Nylon bags of untreated or microwave treated corn were suspended in the rumen of three Holstein steers for 0–48 h, and resulting data were fitted to a non-linear degradation model to calculate effective rumen degradation (ERD). Microwave treatments decreased (P<0.05) the water soluble fraction and increased the potentially degradable fraction of CP, except for 7 min processing time that decreased (P<0.05) the potentially degradable fraction. Processing for 3 and 5 min had no effect, but for 7 min it decreased (P<0.05) ERD of CP. Microwave irradiation for 3 and 5 min increased (P<0.05) the water soluble fraction and decreased the potentially degradable fraction of starch. The degradation rate of the latter fraction increased (P<0.05) with 3 and 5 min processing times. Microwave irradiation for 7 min decreased (P<0.05) the water soluble fraction and the potentially degradable fraction of starch. Processing for 3 and 5 min increased (P<0.05), but for 7 min decreased (P<0.05) ERD of starch. From SDS-PAGE patterns, four major protein components in corn containing prolamin (i.e., zein), albumin, globulin and glutelin fractions were observed. Electrophoretic analysis of protein residues revealed that microwave processing decreased degradation of corn true protein. SDS-PAGE indicated that the bulk of the rumen undegradable protein for untreated corn was zein, while for 5 and 7 min microwave treated corn it was zein and other proteins. In this study, the optimal processing time with 800 W microwave power was judged to be 5 min.

Detection and quantification of protein residues in food grade amino acids and nucleic acids using a dot-blot fluorescent staining method.

Food allergies represent an important health problem in industrialized countries, such that detection and quantitative analysis of the protein considered to be the main allergen is crucial. A dot-blot fluorescent staining method for the detection and quantitative analysis of protein residues in food grade amino acids and nucleic acids is presented. This method combines fluorescence staining with dot-blotting onto PVDF membrane. Several standard proteins, such as bovine serum albumin (66 kDa), lysozyme (14 kDa), ubiquitin (8.6 kDa), bovine insulin (5.7 kDa), and oxidized insulin B chain (3.5 kDa), were detectable at 0.1 ppm using SYPRO Ruby blot stain. Twenty-five different amino acids and two different nucleic acids of food grade were analyzed using this method combined with an internal standard addition method using BSA as an internal standard. All amino acids and nucleic acids were dissolved in 3.6% aqueous HCl and dot-blotted onto PVDF membrane before a large amount of amino acids and nucleic acid were removed. Protein residues and the internal standard protein immobilized on the membrane were stained using SYPRO ruby blot stain. The internal standard in all samples was detectable at 0.1 ppm. Samples were dissolved at 120 or 70 mg/mL, according to their solubility under acidic conditions. The detection limit of protein residues per weight was 0.8-1.4 ppm in amino acids and nucleic acids; residual protein was not detected in any sample.