ABSTRACT A brief summary of the technique of species identifi cation by interrogation of protein ... more ABSTRACT A brief summary of the technique of species identifi cation by interrogation of protein sequence using bottom up proteomics approaches is presented. Instrumentation and software are discussed along with problems of posttranslational, post-mortem and high temperature processing modifi cations to proteins and amino acids that can affect the subsequent interpretation of protein sequence data from high temperature processed meat and bone meal (MBM). Three approaches are described, using a combination of protein purifi cation techniques and mass spectrometry, which can be used to identify animal protein present in MBM through the sequence analysis of the bone protein collagen or the muscle proteins myosin and troponin I.
All stakeholders in cultural heritage share an interest in fabrication methods and material techn... more All stakeholders in cultural heritage share an interest in fabrication methods and material technology. Until now methods for analysis of organic materials, particularly proteins, have not been widely available to researchers at cultural institutions. This paper will describe an analytical method for the identification of collagen-based materials from soft tissue sources and show examples of its application to diverse museum objects. The method, peptide mass fingerprinting (PMF), uses enzymatic digestion of extracted proteins to produce a mixture of peptides. The mass spectrum of the mixture contains characteristic marker ions-a peptide mass fingerprint-which are compared to species-specific markers from references as the basis of identification. Preliminary results indicate that analysis of materials from aged samples, several different tissue types, and tanned or untanned materials yields comparable PMF results. Significantly, PMF is simple, rapid, sensitive and specific, has been implemented in a museum laboratory, and is being practiced successfully by non-specialists.
We applied a recently developed method of collagen peptide mass fingerprinting for taxonomic iden... more We applied a recently developed method of collagen peptide mass fingerprinting for taxonomic identification in faunal remains to 111 specimens from the Neolithic site of Domuztepe in southeastern Turkey. Advances on the published technique allow us to move beyond the key domesticated fauna (sheep, goat, pig and cattle) to include non-domesticates (deer and gazelle, as well as humans). As the animal economy of Domuztepe was dominated by domestic sheep and goats, a large number of the samples tested were morphologically indistinguishable sheep/goat remains. A number of samples represented elements that provide information on age (i.e., mandibles) or sex (pelvis); thus, this represents the first major study to attempt to improve kill-off profiles using collagen fingerprinting. We also show that the collagen extracted from dentine is equally amenable to the proposed methodology as bone collagen.
Determining the extent of overlap between modern humans and other hominins in Eurasia, such as Ne... more Determining the extent of overlap between modern humans and other hominins in Eurasia, such as Neanderthals and Denisovans, is fundamental to understanding the nature of their interactions and what led to the disappearance of archaic hominins. Apart from a possible sporadic pulse recorded in Greece during the Middle Pleistocene, the first settlements of modern humans in Europe have been constrained to ~45,000 to 43,000 years ago. Here, we report hominin fossils from Grotte Mandrin in France that reveal the earliest known presence of modern humans in Europe between 56,800 and 51,700 years ago. This early modern human incursion in the Rhône Valley is associated with technologies unknown in any industry of that age outside Africa or the Levant. Mandrin documents the first alternating occupation of Neanderthals and modern humans, with a modern human fossil and associated Neronian lithic industry found stratigraphically between layers containing Neanderthal remains associated with Mouste...
Fish are a large, highly diverse, and anthropologically important group of vertebrates. However, ... more Fish are a large, highly diverse, and anthropologically important group of vertebrates. However, fish bones are underrepresented in the archaeological literature because they are less stable than those of other taxa and identification of bone to species is often difficult or impossible. We explore a new identification system, ZooMS (Zooarchaeology by Mass Spectrometry), which is based upon protein barcoding. As
Fig. 1. Plot of radiocarbon age versus estimated effective collagen degradation temperature for r... more Fig. 1. Plot of radiocarbon age versus estimated effective collagen degradation temperature for radiocarbon-dated bones from laboratory databases (principally Oxford and Groningen). The line represents the expected calendar age at which 1% of the original collagen remains following a zero-order reaction; almost no bone collagen survives beyond this predicted limit. (Inset) The 99% confidence intervals of amino acid compositions by first two principal component analyses (48% of total variance) for bones from NW Europe aged <11 ky (n = 324), 11 to 110 ky (n = 210), 110 to 130 ky (n = 26), and 130 to 700 ky (n = 31). Pliocene samples are not plotted, as their composition (n = 8) is highly variable and yields of amino acids are low. The orange line indicates a compositional trend observed when compact bone is heated for 32 days at 95°C, which reduces collagen to 1% of the initial concentration [each inflection represents a separate analysis; n = 32)]. The composition becomes more sim...
The unique physiochemical properties of wheat gluten enable a diverse range of food products to b... more The unique physiochemical properties of wheat gluten enable a diverse range of food products to be manufactured. However, gluten triggers coeliac disease, a condition which is treated using a gluten-free diet. Analytical methods are required to confirm if foods are gluten-free, but current immunoassay-based methods can unreliable and proteomic methods offer an alternative but require comprehensive and well annotated sequence databases which are lacking for gluten. A manually a curated database (GluPro V1.0) of gluten proteins, comprising 630 discrete unique full length protein sequences has been compiled. It is representative of the different types of gliadin and glutenin components found in gluten. An in silico comparison of their coeliac toxicity was undertaken by analysing the distribution of coeliac toxic motifs. This demonstrated that whilst the α-gliadin proteins contained more toxic motifs, these were distributed across all gluten protein sub-types. Comparison of annotations observed using a discovery proteomics dataset acquired using ion mobility MS/MS showed that more reliable identifications were obtained using the GluPro V1.0 database compared to the complete reviewed Viridiplantae database. This highlights the value of a curated sequence database specifically designed to support the proteomic workflows and the development of methods to detect and quantify gluten. Significance We have constructed the first manually curated open-source wheat gluten protein sequence database (GluPro V1.0) in a FASTA format to support the application of proteomic methods for gluten protein detection and quantification. We have also analysed the manually verified sequences to give the first comprehensive overview of the distribution of sequences able to elicit a reaction in coeliac disease, the prevalent form of gluten intolerance. Provision of this database will improve the reliability of gluten protein identification by proteomic analysis, and aid the development of targeted mass spectrometry methods in line with Codex Alimentarius Commission requirements for foods designed to meet the needs of gluten intolerant individuals.
Proceedings. Biological sciences / The Royal Society, May 7, 2015
Since the late eighteenth century, fossils of bizarre extinct creatures have been described from ... more Since the late eighteenth century, fossils of bizarre extinct creatures have been described from the Americas, revealing a previously unimagined chapter in the history of mammals. The most bizarre of these are the 'native' South American ungulates thought to represent a group of mammals that evolved in relative isolation on South America, but with an uncertain affinity to any particular placental lineage. Many authors have considered them descended from Laurasian 'condylarths', which also includes the probable ancestors of perissodactyls and artiodactyls, whereas others have placed them either closer to the uniquely South American xenarthrans (anteaters, armadillos and sloths) or the basal afrotherians (e.g. elephants and hyraxes). These hypotheses have been debated owing to conflicting morphological characteristics and the hitherto inability to retrieve molecular information. Of the 'native' South American mammals, only the toxodonts and litopterns persisted...
Asara et al . (Reports, 13 April 2007, p. 280) reported sequencing of Tyrannosaurus rex proteins ... more Asara et al . (Reports, 13 April 2007, p. 280) reported sequencing of Tyrannosaurus rex proteins and used them to establish the evolutionary relationships between birds and dinosaurs. We argue that the reported T. rex peptides may represent statistical artifacts and call for complete data release to enable experimental and computational verification of their findings.
ABSTRACT A brief summary of the technique of species identifi cation by interrogation of protein ... more ABSTRACT A brief summary of the technique of species identifi cation by interrogation of protein sequence using bottom up proteomics approaches is presented. Instrumentation and software are discussed along with problems of posttranslational, post-mortem and high temperature processing modifi cations to proteins and amino acids that can affect the subsequent interpretation of protein sequence data from high temperature processed meat and bone meal (MBM). Three approaches are described, using a combination of protein purifi cation techniques and mass spectrometry, which can be used to identify animal protein present in MBM through the sequence analysis of the bone protein collagen or the muscle proteins myosin and troponin I.
All stakeholders in cultural heritage share an interest in fabrication methods and material techn... more All stakeholders in cultural heritage share an interest in fabrication methods and material technology. Until now methods for analysis of organic materials, particularly proteins, have not been widely available to researchers at cultural institutions. This paper will describe an analytical method for the identification of collagen-based materials from soft tissue sources and show examples of its application to diverse museum objects. The method, peptide mass fingerprinting (PMF), uses enzymatic digestion of extracted proteins to produce a mixture of peptides. The mass spectrum of the mixture contains characteristic marker ions-a peptide mass fingerprint-which are compared to species-specific markers from references as the basis of identification. Preliminary results indicate that analysis of materials from aged samples, several different tissue types, and tanned or untanned materials yields comparable PMF results. Significantly, PMF is simple, rapid, sensitive and specific, has been implemented in a museum laboratory, and is being practiced successfully by non-specialists.
We applied a recently developed method of collagen peptide mass fingerprinting for taxonomic iden... more We applied a recently developed method of collagen peptide mass fingerprinting for taxonomic identification in faunal remains to 111 specimens from the Neolithic site of Domuztepe in southeastern Turkey. Advances on the published technique allow us to move beyond the key domesticated fauna (sheep, goat, pig and cattle) to include non-domesticates (deer and gazelle, as well as humans). As the animal economy of Domuztepe was dominated by domestic sheep and goats, a large number of the samples tested were morphologically indistinguishable sheep/goat remains. A number of samples represented elements that provide information on age (i.e., mandibles) or sex (pelvis); thus, this represents the first major study to attempt to improve kill-off profiles using collagen fingerprinting. We also show that the collagen extracted from dentine is equally amenable to the proposed methodology as bone collagen.
Determining the extent of overlap between modern humans and other hominins in Eurasia, such as Ne... more Determining the extent of overlap between modern humans and other hominins in Eurasia, such as Neanderthals and Denisovans, is fundamental to understanding the nature of their interactions and what led to the disappearance of archaic hominins. Apart from a possible sporadic pulse recorded in Greece during the Middle Pleistocene, the first settlements of modern humans in Europe have been constrained to ~45,000 to 43,000 years ago. Here, we report hominin fossils from Grotte Mandrin in France that reveal the earliest known presence of modern humans in Europe between 56,800 and 51,700 years ago. This early modern human incursion in the Rhône Valley is associated with technologies unknown in any industry of that age outside Africa or the Levant. Mandrin documents the first alternating occupation of Neanderthals and modern humans, with a modern human fossil and associated Neronian lithic industry found stratigraphically between layers containing Neanderthal remains associated with Mouste...
Fish are a large, highly diverse, and anthropologically important group of vertebrates. However, ... more Fish are a large, highly diverse, and anthropologically important group of vertebrates. However, fish bones are underrepresented in the archaeological literature because they are less stable than those of other taxa and identification of bone to species is often difficult or impossible. We explore a new identification system, ZooMS (Zooarchaeology by Mass Spectrometry), which is based upon protein barcoding. As
Fig. 1. Plot of radiocarbon age versus estimated effective collagen degradation temperature for r... more Fig. 1. Plot of radiocarbon age versus estimated effective collagen degradation temperature for radiocarbon-dated bones from laboratory databases (principally Oxford and Groningen). The line represents the expected calendar age at which 1% of the original collagen remains following a zero-order reaction; almost no bone collagen survives beyond this predicted limit. (Inset) The 99% confidence intervals of amino acid compositions by first two principal component analyses (48% of total variance) for bones from NW Europe aged <11 ky (n = 324), 11 to 110 ky (n = 210), 110 to 130 ky (n = 26), and 130 to 700 ky (n = 31). Pliocene samples are not plotted, as their composition (n = 8) is highly variable and yields of amino acids are low. The orange line indicates a compositional trend observed when compact bone is heated for 32 days at 95°C, which reduces collagen to 1% of the initial concentration [each inflection represents a separate analysis; n = 32)]. The composition becomes more sim...
The unique physiochemical properties of wheat gluten enable a diverse range of food products to b... more The unique physiochemical properties of wheat gluten enable a diverse range of food products to be manufactured. However, gluten triggers coeliac disease, a condition which is treated using a gluten-free diet. Analytical methods are required to confirm if foods are gluten-free, but current immunoassay-based methods can unreliable and proteomic methods offer an alternative but require comprehensive and well annotated sequence databases which are lacking for gluten. A manually a curated database (GluPro V1.0) of gluten proteins, comprising 630 discrete unique full length protein sequences has been compiled. It is representative of the different types of gliadin and glutenin components found in gluten. An in silico comparison of their coeliac toxicity was undertaken by analysing the distribution of coeliac toxic motifs. This demonstrated that whilst the α-gliadin proteins contained more toxic motifs, these were distributed across all gluten protein sub-types. Comparison of annotations observed using a discovery proteomics dataset acquired using ion mobility MS/MS showed that more reliable identifications were obtained using the GluPro V1.0 database compared to the complete reviewed Viridiplantae database. This highlights the value of a curated sequence database specifically designed to support the proteomic workflows and the development of methods to detect and quantify gluten. Significance We have constructed the first manually curated open-source wheat gluten protein sequence database (GluPro V1.0) in a FASTA format to support the application of proteomic methods for gluten protein detection and quantification. We have also analysed the manually verified sequences to give the first comprehensive overview of the distribution of sequences able to elicit a reaction in coeliac disease, the prevalent form of gluten intolerance. Provision of this database will improve the reliability of gluten protein identification by proteomic analysis, and aid the development of targeted mass spectrometry methods in line with Codex Alimentarius Commission requirements for foods designed to meet the needs of gluten intolerant individuals.
Proceedings. Biological sciences / The Royal Society, May 7, 2015
Since the late eighteenth century, fossils of bizarre extinct creatures have been described from ... more Since the late eighteenth century, fossils of bizarre extinct creatures have been described from the Americas, revealing a previously unimagined chapter in the history of mammals. The most bizarre of these are the 'native' South American ungulates thought to represent a group of mammals that evolved in relative isolation on South America, but with an uncertain affinity to any particular placental lineage. Many authors have considered them descended from Laurasian 'condylarths', which also includes the probable ancestors of perissodactyls and artiodactyls, whereas others have placed them either closer to the uniquely South American xenarthrans (anteaters, armadillos and sloths) or the basal afrotherians (e.g. elephants and hyraxes). These hypotheses have been debated owing to conflicting morphological characteristics and the hitherto inability to retrieve molecular information. Of the 'native' South American mammals, only the toxodonts and litopterns persisted...
Asara et al . (Reports, 13 April 2007, p. 280) reported sequencing of Tyrannosaurus rex proteins ... more Asara et al . (Reports, 13 April 2007, p. 280) reported sequencing of Tyrannosaurus rex proteins and used them to establish the evolutionary relationships between birds and dinosaurs. We argue that the reported T. rex peptides may represent statistical artifacts and call for complete data release to enable experimental and computational verification of their findings.
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Papers by Mike Buckley