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Papers by Glendon Parker
Journal of Archaeological Science: Reports, Jul 1, 2023
Proceedings of the National Academy of Sciences of the United States of America, Jul 22, 2002
Insulin resistance and  cell toxicity are key features of type 2 diabetes. One leading hypothesi... more Insulin resistance and  cell toxicity are key features of type 2 diabetes. One leading hypothesis suggests that these abnormalities result from excessive flux of nutrients through the UDPhexosamine biosynthetic pathway leading to ''glucose toxicity.'' How the products of the hexosamine pathway mediate these effects is not known. Here, we show that transgenic overexpression of an enzyme using UDP-GlcNAc to modify proteins with O-GlcNAc produces the type 2 diabetic phenotype. Even modest overexpression of an isoform of O-GlcNAc transferase, in muscle and fat, leads to insulin resistance and hyperleptinemia. These data support the proposal that O-linked GlcNAc transferase participates in a hexosamine-dependent signaling pathway that is linked to insulin resistance and leptin production.
In an important breakthrough for the forensic science community, researchers have developed the f... more In an important breakthrough for the forensic science community, researchers have developed the first-ever biological identification method that exploits the information encoded in proteins of human hair.
The FASEB Journal, Apr 1, 2007
Although we have known for many years that most cell surface and extracellular proteins are glyco... more Although we have known for many years that most cell surface and extracellular proteins are glycosylated, only recently have we come to appreciate that most proteins within the nucleus and cytoplasm are also dynamically modified by the addition and removal of saccharides [1]. Indeed, in eukaryotes most polypeptides are glycosylated. Extracellular protein-bound glycans are generally complex and large, whereas cytosolic and nuclear glycans are often modified by simple monosaccharides [2]. Each unique type of protein glycosylation presents special challenges to the structural analyses or identification of glycoproteins by mass spectrometry (MS)[3–9]. MS analyses of extracellular or cell-surface glycoproteins are complicated by the enormous structural diversity of the glycan side chains, by their large size, by the astonishing site-specific structural variability of glycans, and by the fact that many component monosaccharides have the same mass. Mass spectrometric analysis of O-G1cNAc-bearing nuclear and cytoplasmic glycoproteins is confounded by the highly-dynamic nature of the modification [10], causing sub-stoichiometric levels at single sites, by the inherent insensitivity of the method to glycopeptides as compared to unmodified peptides, and most importantly, by the lability of the saccharide linkage under most MS analytical conditions [11, 12]. Nonetheless, mass spectrometry of all types is the most powerful tool currently available to the glycoscientist interested in the structure/functions of posttranslationally modified proteins as they actually occur in biological systems.
Forensic Science International: Genetics Supplement Series, Dec 1, 2017
DNA on touched objects is often present in low amounts, causing poor STR typing results. Testing ... more DNA on touched objects is often present in low amounts, causing poor STR typing results. Testing genetically variable proteins is a promising approach for obtaining additional individualization. We are showing here that it is possible to obtain PCR-STR results for DNA and mass spectrometry protein sequences starting with a single trypsin digestion. For a standard DNA extraction method using Millipore Microcon MW100 filter units, proteinase K was replaced with trypsin, and sodium dodecyl sulfate with ProteaseMAX, a mass spectrometry compatible detergent. This neatly separates both substances: DNA is retained by the membrane with digested proteins in the flow through. It was also possible to amplify trypsin extracted DNA without further purification, but Microcon separation of both fractions was more suitable for both STR and mass spectrometry analysis. When tested in parallel to a standard proteinase K method, the Microcon co-extraction method showed better DNA typing success rates. Mass spectrometry for the Microcon based trypsin co-extraction method yielded a large number of identified proteins, including tissue specific proteins for both fingerprint and saliva samples.
Journal of Forensic Sciences, Mar 20, 2019
Biological evidence analysis from contact traces is adversely affected by low quantity and qualit... more 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.
The 89th Annual Meeting of the American Association of Physical Anthropologists, Los Angeles, CA, 2020
Forensic Science International: Genetics, 2020
Recent reports highlight possible improvements in individual identification using proteomic infor... more Recent reports highlight possible improvements in individual identification using proteomic information from human hair evidence. These reports have stimulated investigation of parameters that affect the utility of proteomic information. In addition to variables already studied relating to processing technique and anatomic origin of hair shafts, an important variable is hair ageing. Present work focuses on the effect of age on protein profiling and analysis of genetically variant peptides (GVPs). Hair protein profiles may be affected by developmental and physiological changes with age of the donor, exposure to different environmental conditions and intrinsic processes, including during storage. First, to explore whether general trends were evident in the population at different ages, hair samples were analyzed from groups of different subjects in their 20's, 40's and 60's. No significant differences were seen as a function of age, but consistent differences were evident between European American and African American hair profiles. Second, samples collected from single individuals at different ages were analyzed. Mostly, these showed few protein expression level differences over periods of 10 years or less, but samples from subjects at 44 and 65 year intervals were distinctly different in profile. The results indicate that use of protein profiling for personal identification, if practical, would be limited to decadal time intervals. Moreover, batch effects were clearly evident in samples processed by different staff. To investigate the contribution of storage (at room temperature) in affecting the outcomes, the same proteomic digests were analyzed for GVPs. In samples stored over 10 years, GVPs were reduced in number in parallel with the yield of identified proteins and unique peptides. However, a very different picture emerged with respect to personal identification. Numbers of GVPs sufficed to distinguish individuals despite the age differences of the samples. As a practical matter, three hair samples per person provided nearly the maximal number obtained from 5 or 6 samples. The random match probability (where the log increased in proportion to the number of GVPs) reached as high as 1 in 10 8. The data indicate that GVP results are dependent on the single nucleotide polymorphism profile of the donor genome, where environmental/processing factors affect only the yield, and thus are consistent despite the ages of the donors and samples and batchwise effects in processing. This conclusion is critical for application to casework where the samples may be in storage for long periods and used to match samples recently collected.
Journal of Archaeological Science, Jun 1, 2021
Abstract Biomolecular sex estimation promises to fill a major gap in the bioarchaeological record... more Abstract Biomolecular sex estimation promises to fill a major gap in the bioarchaeological record by providing estimates of biological sex for skeletal remains with degraded or ambiguous osteological sex-specific markers. Genomic and proteomic sex estimation, like all analytical methods, have limitations and require frameworks to address the problems of low signal samples and the inevitable conflicting results when other methods are used. Proteomic sex estimation is based on the detection of sex-chromosome specific amelogenin protein fragments in enamel using mass spectrometry. Enamel from male individuals contains amelogenin fragments from both the X-and Y-chromosome versions of amelogenin, and enamel from female individuals contains fragments from only the X-chromosome protein. The method is sensitive, robust, quantifiable and reproducible. Researchers have developed, and continue to develop, frameworks to address theoretical problems associated with low levels of detection and conflicting sex estimates that will inevitably occur when multiple methods are used on a sufficiently large dataset. Stamfelj reminds readers that structural variants of the Y-chromosome that delete the amelogenin gene have been detected in forensics and clinical casework. Since this phenomenon would also account for the absence of the AMELY protein in enamel it should therefore be mentioned as an alternative hypothesis by investigators, along with female sex and low peptide signals in mass spectrometry. In his meta-analysis Stamfelj concludes that this is an intrinsic limitation of biomolecular sex estimation, particularly when examining South Asian populations, and should be incorporated in standard analytical sex estimation frameworks. In this comment, we test this assertion by examining the occurrence of AMELY deletion in the systematically sampled, high coverage, large scale, and well-curated populations of the 1000 Genomes Project and Exome Sequencing Project. When using SNP loci in the open reading frame of AMELY, structural deletion was not detected in either project. Confident probabilities of occurrence with associated intervals cannot be determined from null values. We conclude from this that, for now, AMELY deletion should have no bearing on routine biomolecular sex estimation.
The RefSeq Protein Variant Database is a unique protein sequence database, developed for the expr... more The RefSeq Protein Variant Database is a unique protein sequence database, developed for the express purpose of defining variant peptides that can then be detected for use in the identification of individuals. This database is in Mascot compatible FASTA format and can be used in conjunction with proteomic mass spectrometry analytical tools such as X!tandem, Sequest, PEAKs and Mascot.
This thesis was scanned from the print manuscript for digital preservation and is copyright the a... more This thesis was scanned from the print manuscript for digital preservation and is copyright the author. Researchers can access this thesis by asking their local university, institution or public library to make a request on their behalf. Monash staff and postgraduate students can use the link in the References field.
Forensic Science International: Genetics, 2021
This study examines the potential of hair shaft proteomic analysis to delineate genetic relatedne... more This study examines the potential of hair shaft proteomic analysis to delineate genetic relatedness. Proteomic profiling and amino acid sequence analysis provide information for quantitative and statistically-based analysis of individualization and sample similarity. Protein expression levels are a function of cell-specific transcriptional and translational programs. These programs are greatly influenced by an individual's genetic background, and are therefore influenced by familial relatedness as well as ancestry and genetic disease. Proteomic profiles should therefore be more similar among related individuals than unrelated individuals. Likewise, profiles of genetically variant peptides that contain single amino acid polymorphisms, the result of non-synonymous SNP alleles, should behave similarly. The proteomically-inferred SNP alleles should also provide a basis for calculation of combined paternity and sibship indices. We test these hypotheses using matching proteomic and genetic datasets from a family of two adults and four siblings, one of which has a genetic condition that perturbs hair structure and properties. We demonstrate that related individuals, compared to those who are unrelated, have more similar proteomic profiles, profiles of genetically variant peptides and higher combined paternity indices and combined sibship indices. This study builds on previous analyses of hair shaft protein profiling and genetically variant peptide profiles in different real-world scenarios including different human hair shaft body locations and pigmentation status. It also validates the inclusion of proteomic information with other biomolecular substrates in forensic hair shaft analysis, including mitochondrial and nuclear DNA.
Forensic Science International: Genetics, 2021
Protein is a major component of all biological evidence, often the matrix that embeds other biomo... more Protein is a major component of all biological evidence, often the matrix that embeds other biomolecules such as polynucleotides, lipids, carbohydrates, and small molecules. The proteins in a sample reflect the transcriptional and translational program of the originating cell types. Because of this, proteins can be used to identify body fluids and tissues, as well as convey genetic information in the form of single amino acid polymorphisms, the result of non-synonymous SNPs. This review explores the application and potential of forensic proteomics. The historical role that protein analysis played in the development of forensic science is examined. This review details how innovations in proteomic mass spectrometry have addressed many of the historical limitations of forensic protein science, and how the application of forensic proteomics differs from proteomics in the life sciences. Two more developed applications of forensic proteomics are examined in detail: body fluid and tissue identification, and proteomic genotyping. The review then highlights developing areas of proteomics that have the potential to impact forensic science in the near future: fingermark analysis, species identification, peptide toxicology, proteomic sex estimation, and estimation of post-mortem intervals. Finally, the review highlights some of the newer innovations in proteomics that may drive further development of the field. In addition to potential impact, this review also attempts to evaluate the stage of each application in the development, validation and implementation process. This review is targeted at investigators who are interested in learning about proteomics in a forensic context and expanding the amount of information they can extract from biological evidence.
Characterization of ancestry-linked peptide variants in disease-relevant patient tissues represen... more Characterization of ancestry-linked peptide variants in disease-relevant patient tissues represents a foundational step to connect patient ancestry with molecular disease pathogenesis. Nonsynonymous single nucleotide polymorphisms (SNPs) encoding missense substitutions within tryptic peptides exhibiting high allele frequencies in European, African, and East Asian populations, termed peptide ancestry informative markers (pAIMs), were prioritized from 1000 genomes. In silico analysis shows that as few as 20 pAIMs can determine ancestry proportions similarly to >260K SNPs (R2=0.9905). Multiplexed proteomic analysis of >100 human endometrial cancer cell lines and uterine leiomyoma tissues resulted in the quantitation of 62 pAIMs that correlate with self-described race and genotype-confirmed patient ancestry. Candidates include a D451E substitution in GC vitamin D-binding protein previously associated with altered vitamin D levels in African and European populations. These efforts ...
Journal of Forensic Sciences, 2019
Biological evidence analysis from contact traces is adversely affected by low quantity and qualit... more 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 AE 1.87 ng and swabs, 1.34 AE 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 AE 10.9, (tape) versus 47.9 AE 20.4 (swabs). Proteins can be collected from fingerprints at levels necessary to provide identifying information, thus expanding information obtained from challenging evidence.
Journal of Archaeological Science, 2021
Abstract Biomolecular sex estimation promises to fill a major gap in the bioarchaeological record... more Abstract Biomolecular sex estimation promises to fill a major gap in the bioarchaeological record by providing estimates of biological sex for skeletal remains with degraded or ambiguous osteological sex-specific markers. Genomic and proteomic sex estimation, like all analytical methods, have limitations and require frameworks to address the problems of low signal samples and the inevitable conflicting results when other methods are used. Proteomic sex estimation is based on the detection of sex-chromosome specific amelogenin protein fragments in enamel using mass spectrometry. Enamel from male individuals contains amelogenin fragments from both the X-and Y-chromosome versions of amelogenin, and enamel from female individuals contains fragments from only the X-chromosome protein. The method is sensitive, robust, quantifiable and reproducible. Researchers have developed, and continue to develop, frameworks to address theoretical problems associated with low levels of detection and conflicting sex estimates that will inevitably occur when multiple methods are used on a sufficiently large dataset. Stamfelj reminds readers that structural variants of the Y-chromosome that delete the amelogenin gene have been detected in forensics and clinical casework. Since this phenomenon would also account for the absence of the AMELY protein in enamel it should therefore be mentioned as an alternative hypothesis by investigators, along with female sex and low peptide signals in mass spectrometry. In his meta-analysis Stamfelj concludes that this is an intrinsic limitation of biomolecular sex estimation, particularly when examining South Asian populations, and should be incorporated in standard analytical sex estimation frameworks. In this comment, we test this assertion by examining the occurrence of AMELY deletion in the systematically sampled, high coverage, large scale, and well-curated populations of the 1000 Genomes Project and Exome Sequencing Project. When using SNP loci in the open reading frame of AMELY, structural deletion was not detected in either project. Confident probabilities of occurrence with associated intervals cannot be determined from null values. We conclude from this that, for now, AMELY deletion should have no bearing on routine biomolecular sex estimation.
<p><b>A</b>) Genetically variant peptides (GVPs) that contained single amino-ac... more <p><b>A</b>) Genetically variant peptides (GVPs) that contained single amino-acid polymorphisms (SAPs) were identified in both European-American cohorts (EA1 and EA2) and collated for each subject. Imputed nsSNP alleles (Gene Name = GN, SNP accession number = rs#, allele nucleotide = nuc) were directly compared to the genotype resulting from direct Sanger sequencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160653#pone.0160653.s011" target="_blank">S1 Methods</a>). Correctly imputed nsSNP alleles (TP, true positives) are indicated by a blue square. Imputed alleles that were incorrectly predicted (FP, false positive) are indicated by red squares. Alleles that were identified using Sanger sequencing, but did not contain a resulting GVP in the matching proteomic dataset (FN, false negative) are indicated by light green squares. Alleles absent in both subjects DNA and in resulting proteomic datasets (TN, true negatives) are indicated by white squares[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160653#pone.0160653.ref049" target="_blank">49</a>]. Failed Sanger sequencing determination of nsSNP allelic status is indicated by grey. <b>B</b>) The effectiveness of each SAP-containing peptide to impute nsSNP alleles was also quantified. The sensitivity of each genetically variant peptide, measured as the proportion of nsSNP-alleles that are correctly detected and imputed (TP/(TP+FN)), was calculated as a percentage (log<sub>10</sub>(%). The positive predictive value (PPV) of genetically variant peptide-based SNP imputations was calculated as the percentage of correct validated SNP imputations of all imputations (TP/(TP + FP); log<sub>10</sub>(%))[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160653#pone.0160653.ref049" target="_blank">49</a>]. <b>C</b>)</p
Journal of Archaeological Science: Reports, Jul 1, 2023
Proceedings of the National Academy of Sciences of the United States of America, Jul 22, 2002
Insulin resistance and  cell toxicity are key features of type 2 diabetes. One leading hypothesi... more Insulin resistance and  cell toxicity are key features of type 2 diabetes. One leading hypothesis suggests that these abnormalities result from excessive flux of nutrients through the UDPhexosamine biosynthetic pathway leading to ''glucose toxicity.'' How the products of the hexosamine pathway mediate these effects is not known. Here, we show that transgenic overexpression of an enzyme using UDP-GlcNAc to modify proteins with O-GlcNAc produces the type 2 diabetic phenotype. Even modest overexpression of an isoform of O-GlcNAc transferase, in muscle and fat, leads to insulin resistance and hyperleptinemia. These data support the proposal that O-linked GlcNAc transferase participates in a hexosamine-dependent signaling pathway that is linked to insulin resistance and leptin production.
In an important breakthrough for the forensic science community, researchers have developed the f... more In an important breakthrough for the forensic science community, researchers have developed the first-ever biological identification method that exploits the information encoded in proteins of human hair.
The FASEB Journal, Apr 1, 2007
Although we have known for many years that most cell surface and extracellular proteins are glyco... more Although we have known for many years that most cell surface and extracellular proteins are glycosylated, only recently have we come to appreciate that most proteins within the nucleus and cytoplasm are also dynamically modified by the addition and removal of saccharides [1]. Indeed, in eukaryotes most polypeptides are glycosylated. Extracellular protein-bound glycans are generally complex and large, whereas cytosolic and nuclear glycans are often modified by simple monosaccharides [2]. Each unique type of protein glycosylation presents special challenges to the structural analyses or identification of glycoproteins by mass spectrometry (MS)[3–9]. MS analyses of extracellular or cell-surface glycoproteins are complicated by the enormous structural diversity of the glycan side chains, by their large size, by the astonishing site-specific structural variability of glycans, and by the fact that many component monosaccharides have the same mass. Mass spectrometric analysis of O-G1cNAc-bearing nuclear and cytoplasmic glycoproteins is confounded by the highly-dynamic nature of the modification [10], causing sub-stoichiometric levels at single sites, by the inherent insensitivity of the method to glycopeptides as compared to unmodified peptides, and most importantly, by the lability of the saccharide linkage under most MS analytical conditions [11, 12]. Nonetheless, mass spectrometry of all types is the most powerful tool currently available to the glycoscientist interested in the structure/functions of posttranslationally modified proteins as they actually occur in biological systems.
Forensic Science International: Genetics Supplement Series, Dec 1, 2017
DNA on touched objects is often present in low amounts, causing poor STR typing results. Testing ... more DNA on touched objects is often present in low amounts, causing poor STR typing results. Testing genetically variable proteins is a promising approach for obtaining additional individualization. We are showing here that it is possible to obtain PCR-STR results for DNA and mass spectrometry protein sequences starting with a single trypsin digestion. For a standard DNA extraction method using Millipore Microcon MW100 filter units, proteinase K was replaced with trypsin, and sodium dodecyl sulfate with ProteaseMAX, a mass spectrometry compatible detergent. This neatly separates both substances: DNA is retained by the membrane with digested proteins in the flow through. It was also possible to amplify trypsin extracted DNA without further purification, but Microcon separation of both fractions was more suitable for both STR and mass spectrometry analysis. When tested in parallel to a standard proteinase K method, the Microcon co-extraction method showed better DNA typing success rates. Mass spectrometry for the Microcon based trypsin co-extraction method yielded a large number of identified proteins, including tissue specific proteins for both fingerprint and saliva samples.
Journal of Forensic Sciences, Mar 20, 2019
Biological evidence analysis from contact traces is adversely affected by low quantity and qualit... more 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.
The 89th Annual Meeting of the American Association of Physical Anthropologists, Los Angeles, CA, 2020
Forensic Science International: Genetics, 2020
Recent reports highlight possible improvements in individual identification using proteomic infor... more Recent reports highlight possible improvements in individual identification using proteomic information from human hair evidence. These reports have stimulated investigation of parameters that affect the utility of proteomic information. In addition to variables already studied relating to processing technique and anatomic origin of hair shafts, an important variable is hair ageing. Present work focuses on the effect of age on protein profiling and analysis of genetically variant peptides (GVPs). Hair protein profiles may be affected by developmental and physiological changes with age of the donor, exposure to different environmental conditions and intrinsic processes, including during storage. First, to explore whether general trends were evident in the population at different ages, hair samples were analyzed from groups of different subjects in their 20's, 40's and 60's. No significant differences were seen as a function of age, but consistent differences were evident between European American and African American hair profiles. Second, samples collected from single individuals at different ages were analyzed. Mostly, these showed few protein expression level differences over periods of 10 years or less, but samples from subjects at 44 and 65 year intervals were distinctly different in profile. The results indicate that use of protein profiling for personal identification, if practical, would be limited to decadal time intervals. Moreover, batch effects were clearly evident in samples processed by different staff. To investigate the contribution of storage (at room temperature) in affecting the outcomes, the same proteomic digests were analyzed for GVPs. In samples stored over 10 years, GVPs were reduced in number in parallel with the yield of identified proteins and unique peptides. However, a very different picture emerged with respect to personal identification. Numbers of GVPs sufficed to distinguish individuals despite the age differences of the samples. As a practical matter, three hair samples per person provided nearly the maximal number obtained from 5 or 6 samples. The random match probability (where the log increased in proportion to the number of GVPs) reached as high as 1 in 10 8. The data indicate that GVP results are dependent on the single nucleotide polymorphism profile of the donor genome, where environmental/processing factors affect only the yield, and thus are consistent despite the ages of the donors and samples and batchwise effects in processing. This conclusion is critical for application to casework where the samples may be in storage for long periods and used to match samples recently collected.
Journal of Archaeological Science, Jun 1, 2021
Abstract Biomolecular sex estimation promises to fill a major gap in the bioarchaeological record... more Abstract Biomolecular sex estimation promises to fill a major gap in the bioarchaeological record by providing estimates of biological sex for skeletal remains with degraded or ambiguous osteological sex-specific markers. Genomic and proteomic sex estimation, like all analytical methods, have limitations and require frameworks to address the problems of low signal samples and the inevitable conflicting results when other methods are used. Proteomic sex estimation is based on the detection of sex-chromosome specific amelogenin protein fragments in enamel using mass spectrometry. Enamel from male individuals contains amelogenin fragments from both the X-and Y-chromosome versions of amelogenin, and enamel from female individuals contains fragments from only the X-chromosome protein. The method is sensitive, robust, quantifiable and reproducible. Researchers have developed, and continue to develop, frameworks to address theoretical problems associated with low levels of detection and conflicting sex estimates that will inevitably occur when multiple methods are used on a sufficiently large dataset. Stamfelj reminds readers that structural variants of the Y-chromosome that delete the amelogenin gene have been detected in forensics and clinical casework. Since this phenomenon would also account for the absence of the AMELY protein in enamel it should therefore be mentioned as an alternative hypothesis by investigators, along with female sex and low peptide signals in mass spectrometry. In his meta-analysis Stamfelj concludes that this is an intrinsic limitation of biomolecular sex estimation, particularly when examining South Asian populations, and should be incorporated in standard analytical sex estimation frameworks. In this comment, we test this assertion by examining the occurrence of AMELY deletion in the systematically sampled, high coverage, large scale, and well-curated populations of the 1000 Genomes Project and Exome Sequencing Project. When using SNP loci in the open reading frame of AMELY, structural deletion was not detected in either project. Confident probabilities of occurrence with associated intervals cannot be determined from null values. We conclude from this that, for now, AMELY deletion should have no bearing on routine biomolecular sex estimation.
The RefSeq Protein Variant Database is a unique protein sequence database, developed for the expr... more The RefSeq Protein Variant Database is a unique protein sequence database, developed for the express purpose of defining variant peptides that can then be detected for use in the identification of individuals. This database is in Mascot compatible FASTA format and can be used in conjunction with proteomic mass spectrometry analytical tools such as X!tandem, Sequest, PEAKs and Mascot.
This thesis was scanned from the print manuscript for digital preservation and is copyright the a... more This thesis was scanned from the print manuscript for digital preservation and is copyright the author. Researchers can access this thesis by asking their local university, institution or public library to make a request on their behalf. Monash staff and postgraduate students can use the link in the References field.
Forensic Science International: Genetics, 2021
This study examines the potential of hair shaft proteomic analysis to delineate genetic relatedne... more This study examines the potential of hair shaft proteomic analysis to delineate genetic relatedness. Proteomic profiling and amino acid sequence analysis provide information for quantitative and statistically-based analysis of individualization and sample similarity. Protein expression levels are a function of cell-specific transcriptional and translational programs. These programs are greatly influenced by an individual's genetic background, and are therefore influenced by familial relatedness as well as ancestry and genetic disease. Proteomic profiles should therefore be more similar among related individuals than unrelated individuals. Likewise, profiles of genetically variant peptides that contain single amino acid polymorphisms, the result of non-synonymous SNP alleles, should behave similarly. The proteomically-inferred SNP alleles should also provide a basis for calculation of combined paternity and sibship indices. We test these hypotheses using matching proteomic and genetic datasets from a family of two adults and four siblings, one of which has a genetic condition that perturbs hair structure and properties. We demonstrate that related individuals, compared to those who are unrelated, have more similar proteomic profiles, profiles of genetically variant peptides and higher combined paternity indices and combined sibship indices. This study builds on previous analyses of hair shaft protein profiling and genetically variant peptide profiles in different real-world scenarios including different human hair shaft body locations and pigmentation status. It also validates the inclusion of proteomic information with other biomolecular substrates in forensic hair shaft analysis, including mitochondrial and nuclear DNA.
Forensic Science International: Genetics, 2021
Protein is a major component of all biological evidence, often the matrix that embeds other biomo... more Protein is a major component of all biological evidence, often the matrix that embeds other biomolecules such as polynucleotides, lipids, carbohydrates, and small molecules. The proteins in a sample reflect the transcriptional and translational program of the originating cell types. Because of this, proteins can be used to identify body fluids and tissues, as well as convey genetic information in the form of single amino acid polymorphisms, the result of non-synonymous SNPs. This review explores the application and potential of forensic proteomics. The historical role that protein analysis played in the development of forensic science is examined. This review details how innovations in proteomic mass spectrometry have addressed many of the historical limitations of forensic protein science, and how the application of forensic proteomics differs from proteomics in the life sciences. Two more developed applications of forensic proteomics are examined in detail: body fluid and tissue identification, and proteomic genotyping. The review then highlights developing areas of proteomics that have the potential to impact forensic science in the near future: fingermark analysis, species identification, peptide toxicology, proteomic sex estimation, and estimation of post-mortem intervals. Finally, the review highlights some of the newer innovations in proteomics that may drive further development of the field. In addition to potential impact, this review also attempts to evaluate the stage of each application in the development, validation and implementation process. This review is targeted at investigators who are interested in learning about proteomics in a forensic context and expanding the amount of information they can extract from biological evidence.
Characterization of ancestry-linked peptide variants in disease-relevant patient tissues represen... more Characterization of ancestry-linked peptide variants in disease-relevant patient tissues represents a foundational step to connect patient ancestry with molecular disease pathogenesis. Nonsynonymous single nucleotide polymorphisms (SNPs) encoding missense substitutions within tryptic peptides exhibiting high allele frequencies in European, African, and East Asian populations, termed peptide ancestry informative markers (pAIMs), were prioritized from 1000 genomes. In silico analysis shows that as few as 20 pAIMs can determine ancestry proportions similarly to >260K SNPs (R2=0.9905). Multiplexed proteomic analysis of >100 human endometrial cancer cell lines and uterine leiomyoma tissues resulted in the quantitation of 62 pAIMs that correlate with self-described race and genotype-confirmed patient ancestry. Candidates include a D451E substitution in GC vitamin D-binding protein previously associated with altered vitamin D levels in African and European populations. These efforts ...
Journal of Forensic Sciences, 2019
Biological evidence analysis from contact traces is adversely affected by low quantity and qualit... more 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 AE 1.87 ng and swabs, 1.34 AE 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 AE 10.9, (tape) versus 47.9 AE 20.4 (swabs). Proteins can be collected from fingerprints at levels necessary to provide identifying information, thus expanding information obtained from challenging evidence.
Journal of Archaeological Science, 2021
Abstract Biomolecular sex estimation promises to fill a major gap in the bioarchaeological record... more Abstract Biomolecular sex estimation promises to fill a major gap in the bioarchaeological record by providing estimates of biological sex for skeletal remains with degraded or ambiguous osteological sex-specific markers. Genomic and proteomic sex estimation, like all analytical methods, have limitations and require frameworks to address the problems of low signal samples and the inevitable conflicting results when other methods are used. Proteomic sex estimation is based on the detection of sex-chromosome specific amelogenin protein fragments in enamel using mass spectrometry. Enamel from male individuals contains amelogenin fragments from both the X-and Y-chromosome versions of amelogenin, and enamel from female individuals contains fragments from only the X-chromosome protein. The method is sensitive, robust, quantifiable and reproducible. Researchers have developed, and continue to develop, frameworks to address theoretical problems associated with low levels of detection and conflicting sex estimates that will inevitably occur when multiple methods are used on a sufficiently large dataset. Stamfelj reminds readers that structural variants of the Y-chromosome that delete the amelogenin gene have been detected in forensics and clinical casework. Since this phenomenon would also account for the absence of the AMELY protein in enamel it should therefore be mentioned as an alternative hypothesis by investigators, along with female sex and low peptide signals in mass spectrometry. In his meta-analysis Stamfelj concludes that this is an intrinsic limitation of biomolecular sex estimation, particularly when examining South Asian populations, and should be incorporated in standard analytical sex estimation frameworks. In this comment, we test this assertion by examining the occurrence of AMELY deletion in the systematically sampled, high coverage, large scale, and well-curated populations of the 1000 Genomes Project and Exome Sequencing Project. When using SNP loci in the open reading frame of AMELY, structural deletion was not detected in either project. Confident probabilities of occurrence with associated intervals cannot be determined from null values. We conclude from this that, for now, AMELY deletion should have no bearing on routine biomolecular sex estimation.
<p><b>A</b>) Genetically variant peptides (GVPs) that contained single amino-ac... more <p><b>A</b>) Genetically variant peptides (GVPs) that contained single amino-acid polymorphisms (SAPs) were identified in both European-American cohorts (EA1 and EA2) and collated for each subject. Imputed nsSNP alleles (Gene Name = GN, SNP accession number = rs#, allele nucleotide = nuc) were directly compared to the genotype resulting from direct Sanger sequencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160653#pone.0160653.s011" target="_blank">S1 Methods</a>). Correctly imputed nsSNP alleles (TP, true positives) are indicated by a blue square. Imputed alleles that were incorrectly predicted (FP, false positive) are indicated by red squares. Alleles that were identified using Sanger sequencing, but did not contain a resulting GVP in the matching proteomic dataset (FN, false negative) are indicated by light green squares. Alleles absent in both subjects DNA and in resulting proteomic datasets (TN, true negatives) are indicated by white squares[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160653#pone.0160653.ref049" target="_blank">49</a>]. Failed Sanger sequencing determination of nsSNP allelic status is indicated by grey. <b>B</b>) The effectiveness of each SAP-containing peptide to impute nsSNP alleles was also quantified. The sensitivity of each genetically variant peptide, measured as the proportion of nsSNP-alleles that are correctly detected and imputed (TP/(TP+FN)), was calculated as a percentage (log<sub>10</sub>(%). The positive predictive value (PPV) of genetically variant peptide-based SNP imputations was calculated as the percentage of correct validated SNP imputations of all imputations (TP/(TP + FP); log<sub>10</sub>(%))[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160653#pone.0160653.ref049" target="_blank">49</a>]. <b>C</b>)</p
Science Reports, Hature, 2020
Sex estimation of skeletons is fundamental to many archaeological studies. currently, three appro... more Sex estimation of skeletons is fundamental to many archaeological studies. currently, three approaches are available to estimate sex-osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. We present matching osteological, shotgun-genomic, and proteomic data to estimate the sex of 55 individuals, each with an independent radiocarbon date between 2,440 and 100 cal BP, from two ancestral Ohlone sites in Central California. Sex estimation was possible in 100% of this burial sample using proteomics, in 91% using genomics, and in 51% using osteology. Agreement between the methods was high, however conflicts did occur. Genomic sex estimates were 100% consistent with proteomic and osteological estimates when DNA reads were above 100,000 total sequences. However, more than half the samples had DNA read numbers below this threshold, producing high rates of conflict with osteological and proteomic data where nine out of twenty conditional DNA sex estimates conflicted with proteomics. While the DNA signal decreased by an order of magnitude in the older burial samples, there was no decrease in proteomic signal. We conclude that proteomics provides an important complement to osteological and shotgun-genomic sex estimation.