Peptide ancestry informative markers in uterine neoplasms from women of European, African, and Asian ancestry.

Characterization of ancestry-linked peptide variants in disease-relevant patient tissues represents a foundational step to connect patient ancestry with disease pathogenesis. Nonsynonymous single-nucleotide polymorphisms 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 identified that as few as 20 pAIMs can determine ancestry proportions similarly to >260K SNPs (R2 = 0.99). Multiplexed proteomic analysis of >100 human endometrial cancer cell lines and uterine leiomyoma tissues combined resulted in the quantitation of 62 pAIMs that correlate with patient race and genotype-confirmed ancestry. Candidates include a D451E substitution in GC vitamin D-binding protein previously associated with altered vitamin D levels in African and European populations. pAIMs will support generalized proteoancestry assessment as well as efforts investigating the impact of ancestry on the human proteome and how this relates to the pathogenesis of uterine neoplasms.

Alternative LC-MS/MS Platforms and Data Acquisition Strategies for Proteomic Genotyping of Human Hair Shafts.

Protein is a major component of all biological evidence. Proteomic genotyping is the use of genetically variant peptides (GVPs) that contain single-amino-acid polymorphisms to infer the genotype of matching nonsynonymous single-nucleotide polymorphisms for the individual from whom the protein sample originated. This can be used to statistically associate an individual to evidence found at a crime scene. The utility of the inferred genotype increases as the detection of GVPs increases, which is the direct result of technology transfer to mass spectrometry platforms typically available. Digests of single (2 cm) human hair shafts from three European and two African subjects were analyzed using data-dependent acquisition on a Q-Exactive Plus Hybrid Quadrupole-Orbitrap system, data-independent acquisition and a variant of parallel reaction monitoring (PRM) on an Orbitrap Fusion Lumos Tribrid system, and multiple reaction monitoring (MRM) on an Agilent 6495 triple quadrupole system. In our hands, average GVP detection from a selected panel of 24 GVPs increased from 6.5 ± 1.1 and 3.1 ± 0.8 using data-dependent and -independent acquisition to 9.5 ± 0.7 and 11.7 ± 1.7 using PRM and MRM (p < 0.05), respectively. PRM resulted in a 1.3-fold increase in detection sensitivity, and MRM resulted in a 1.6-fold increase in detection sensitivity. This increase in biomarker detection has a functional impact on the statistical association of a protein sample and an individual. Increased biomarker sensitivity, using Markov Chain Monte Carlo modeling, produced a median-estimated random match probability of over 1 in 10 trillion from a single hair using targeted proteomics. For PRM and MRM, detected GVPs were validated by the inclusion of stable isotope-labeled peptides in each sample, which served also as a detection trigger. This research accomplishes two aims: the demonstration of utility for alternative analytical platforms in proteomic genotyping and the establishment of validation methods for the evaluation of inferred genotypes.

Elucidation of familial relationships using hair shaft proteomics.

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 proteomics.

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.

Female hunters of the early Americas.

Sexual division of labor with females as gatherers and males as hunters is a major empirical regularity of hunter-gatherer ethnography, suggesting an ancestral behavioral pattern. We present an archeological discovery and meta-analysis that challenge the man-the-hunter hypothesis. Excavations at the Andean highland site of Wilamaya Patjxa reveal a 9000-year-old human burial (WMP6) associated with a hunting toolkit of stone projectile points and animal processing tools. Osteological, proteomic, and isotopic analyses indicate that this early hunter was a young adult female who subsisted on terrestrial plants and animals. Analysis of Late Pleistocene and Early Holocene burial practices throughout the Americas situate WMP6 as the earliest and most secure hunter burial in a sample that includes 10 other females in statistical parity with early male hunter burials. The findings are consistent with nongendered labor practices in which early hunter-gatherer females were big-game hunters.

A comparison of proteomic, genomic, and osteological methods of archaeological sex estimation.

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.

Age-Related Changes in Hair Shaft Protein Profiling and Genetically Variant Peptides.

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 108. 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.

Optimal processing for proteomic genotyping of single human hairs.

The use of hair evidence for human identification is undergoing considerable improvement through the adoption of proteomic genotyping. Unlike traditional microscopic comparisons, protein sequencing provides quantitative and empirically based estimates for random match probability. Non-synonymous SNPs are translated as single amino acid polymorphisms and result in genetically variant peptides. Using high resolution mass spectrometry, these peptides can be detected in hair shaft proteins and used to infer the genotypes of corresponding SNP alleles. We describe experiments to optimize the proteomic genotyping approach to individual identification from a single human scalp hair 2 cm in length (∼100 µg). This is a necessary step to develop a protocol that will be useful to forensic investigators. To increase peptide yield from hair, and to maximize genetically variant peptide and ancestral information, we examined the conditions for reduction, alkylation, and protein digestion that specifically address the distinctive chemistry of the hair shaft. Results indicate that optimal conditions for proteomic analysis of a single human hair include 6 h of reduction with 100 mM dithiothreitol at room temperature, alkylation with 200 mM iodoacetamide for 45 min, and 6 h of digestion with two 1:50 (enzyme:protein) additions of stabilized trypsin at room temperature, with stirring incorporated into all three steps. Our final conditions using optimized temperatures and incubation times increased the average number of genetically variant peptides from 20 ±â€¯5 to 73 ±â€¯5 (p = 1 × 10-13), excluding intractable hair samples. Random match probabilities reached up to 1 in 620 million from a single hair with a median value of 1 in 1.1 million, compared to a maximum random match probability of 1 in 1380 and a median value of 1 in 24 for the original hair protein extraction method. Ancestral information was also present in the data. While the number of genetically variant peptides detected were equivalent for both European and African subjects, the estimated random match probabilities for inferred genotypes of European subjects were considerably smaller in African reference populations and vice versa, resulting in a difference in likelihood ratios of 6.8 orders of magnitude. This research will assure uniformity in results across different biogeographic backgrounds and enhance the use of novel peptide analysis in forensic science by helping to optimize genetically variant peptide yields and discovery. This work also introduces two algorithms, GVP Finder and GVP Scout, which facilitate searches, calculate random match probabilities, and aid in discovery of genetically variant peptides.

Proteomic genotyping: Using mass spectrometry to infer SNP genotypes in pigmented and non-pigmented hair.

Proteomic genotyping uses genetically variant peptides that contain single amino acid polymorphisms to infer the genotype of corresponding non-synonymous SNP alleles. We have focused on hair proteins as a source of protein-based genetic information in a forensic context. An optimized sample processing protocol for hair shafts has been developed for use on a single hair that allows us to conduct validation protocols on real world samples. This includes whether the inferred SNP genotypes are robust and not systematically affected by biological or chemical variation in hair proteomes that might be obtained from a crime scene. To this end we analyzed the hair of 4 mature individuals with a mixture of pigmented and non-pigmented hair. We demonstrate significant changes in the proteomes of grey versus pigmented hair. Vesicle specific proteins and lipid catabolism proteins were enriched in pigmented hair, and housekeeping proteins and lipid anabolic enzymes were enriched in grey, non-pigmented hair. The resulting profiles of genetically variant peptides, however, were more correlated with profiles from the same individuals regardless of pigmentation status. Together with other published evidence, this finding indicates that profiles of genetically variant peptides are robust and more correlated with other genetically variant peptide profiles from the same individual irrespective of changes occurring in the hair protein profile. Based on this small sample, investigators using profiles of genetically variant peptides to infer random match probabilities should not expect to observe differences based on the pigmentation of the hair shaft.

Proteomic genotyping of fingermark donors with genetically variant peptides.

Proteomic genotyping detects single amino acid polymorphisms to infer the genotype of corresponding non-synonymous SNPs. Like any DNA genotype, these inferences can be used to estimate random match probability. Fingermarks are a common source of biological evidence that is sample limited and a highly variable source of identifying DNA. Genetically variant peptides from fingermarks, that contain single amino acid polymorphisms, are an additional source of identifying genetic information. To discover these peptide biomarkers epidermal corneocytes from 9 subjects were isolated, processed, digested with trypsin and applied to mass spectrometry. The resulting proteomic and matching exome datasets were used to discover, characterize and validate 60 genetically variant peptides. An average of 28.8 ± 4.4 genetically variant peptides were detected from each subject resulting in a total of 264 SNP allele inferences with 260 true and 4 false positives, a false discovery rate of 1.5%. Random match probabilities were estimated using the genotype frequencies from the matching major populations in the 1000 Genomes Project. Estimates ranged up to a value of 1 in 1.7 × 108, with a median probability of 1 in 2.4 × 106. Furthermore, the proteomically-inferred genotypes are likely to be compatible with the STR-based random match probability estimates since the closest STR locus was 2.2 Mb from the nearest GVP-inferred SNP. This project represents a novel mode of genetic information that can be obtained from fingermarks and has the potential to complement other methods of human identification including analysis of ridge patterns or touch DNA.

Comparison of protein expression levels and proteomically-inferred genotypes using human hair from different body sites.

The microanatomy of human hair differs as a function of the site of origin on the body. This was a major consideration when anatomical features of hair were used as a means of comparison and human identification. Recent advances have demonstrated that proteomics of the hair shaft can be used to develop profiles of protein abundance and genetically variant peptides, the latter in turn being used to infer genotypes of SNP alleles. Because the profile of proteins would be expected to change as hair anatomy changes, it is an open question if the profile of genetically variant peptides will also change. While some sample to sample variation is expected, a potential drawback of using genetically variant peptides to infer an individual genotype is that the proteomic profile might change as a function of body site origin as well as an individual's genotype. The hypothesis in this study is that the profile of hair shaft genetically variant peptides depends more on an individual's genotype than on the site of hair shaft origin. To test this an analysis of both protein expression levels and genetically variant peptides was conducted on 4 body sites (scalp, axillary, beard and pubic hair) from 5 individuals with 4 biological replicates. Levels of protein expression were estimated using label-free quantification on resulting proteomic mass spectrometry datasets. The same datasets were then also analyzed for the presence of genetically variant peptides. This study demonstrates that the protein profiles of hair shafts varied as a function of somatic origin. By contrast the profile of genetically variant peptides, and resulting inferred genotype of SNP alleles, were more dependent on the individual. In this study random match probabilities ranged up to 1 in 196. Individual identification based on genetically variant peptides therefore can be obtained from human hair without regard to the site of origin. If the site of hair shaft origin was legally relevant then microscopic analysis is still necessary. This study demonstrates the utility of proteomic analysis for extracting forensic information from hair shaft evidence.

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

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

Protein-based forensic identification using genetically variant peptides in human bone.

Bone tissue contains organic material that is useful for forensic investigations and may contain preserved endogenous protein that can persist in the environment for extended periods of time over a range of conditions. Single amino acid polymorphisms in these proteins reflect genetic information since they result from non-synonymous single nucleotide polymorphisms (SNPs) in DNA. Detection of genetically variant peptides (GVPs) - those peptides that contain amino acid polymorphisms - in digests of bone proteins allows for the corresponding SNP alleles to be inferred. Resulting genetic profiles can be used to calculate statistical measures of association between a bone sample and an individual. In this study proteomic analysis on rib cortical bone samples from 10 recently deceased individuals demonstrates this concept. A straight-forward acidic demineralization protocol yielded proteins that were digested with trypsin. Tryptic digests were analyzed by liquid chromatography mass spectrometry. A total of 1736 different proteins were identified across all resulting datasets. On average, individual samples contained 454±121 (x¯±σ) proteins. Thirty-five genetically variant peptides were identified from 15 observed proteins. Overall, 134 SNP inferences were made based on proteomically detected GVPs, which were confirmed by sequencing of subject DNA. Inferred individual SNP genetic profiles ranged in random match probability (RMP) from 1/6 to 1/42,472 when calculated with European population frequencies in the 1000 Genomes Project, Phase 3. Similarly, RMPs based on African population frequencies were calculated for each SNP genetic profile and likelihood ratios (LR) were obtained by dividing each European RMP by the corresponding African RMP. Resulting LR values ranged from 1.4 to 825 with a median value of 16. GVP markers offer a basis for the identification of compromised skeletal remains independent of the presence of DNA template.

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

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

Demonstration of Protein-Based Human Identification Using the Hair Shaft Proteome.

Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects' DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European-American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.

The 3T3-L1 adipocyte glycogen proteome.

BACKGROUND: Glycogen is a branched polysaccharide of glucose residues, consisting of α-1-4 glycosidic linkages with α-1-6 branches that together form multi-layered particles ranging in size from 30 nm to 300 nm. Glycogen spatial conformation and intracellular organization are highly regulated processes. Glycogen particles interact with their metabolizing enzymes and are associated with a variety of proteins that intervene in its biology, controlling its structure, particle size and sub-cellular distribution. The function of glycogen in adipose tissue is not well understood but appears to have a pivotal role as a regulatory mechanism informing the cells on substrate availability for triacylglycerol synthesis. To provide new molecular insights into the role of adipocyte glycogen we analyzed the glycogen-associated proteome from differentiated 3T3-L1-adipocytes. RESULTS: Glycogen particles from 3T3-L1-adipocytes were purified using a series of centrifugation steps followed by specific elution of glycogen bound proteins using α-1,4 glucose oligosaccharides, or maltodextrins, and tandem mass spectrometry. We identified regulatory proteins, 14-3-3 proteins, RACK1 and protein phosphatase 1 glycogen targeting subunit 3D. Evidence was also obtained for a regulated subcellular distribution of the glycogen particle: metabolic and mitochondrial proteins were abundant. Unlike the recently analyzed hepatic glycogen proteome, no endoplasmic proteins were detected, along with the recently described starch-binding domain protein 1. Other regulatory proteins which have previously been described as glycogen-associated proteins were not detected, including laforin, the AMPK beta-subunit and protein targeting to glycogen (PTG). CONCLUSIONS: These data provide new molecular insights into the regulation of glycogen-bound proteins that are associated with the maintenance, organization and localization of the adipocyte glycogen particle.

NANOGP8: evolution of a human-specific retro-oncogene.

NANOGP8 is a human (Homo sapiens) retrogene, expressed predominantly in cancer cells where its protein product is tumorigenic. It arose through retrotransposition from its parent gene, NANOG, which is expressed predominantly in embryonic stem cells. Based on identification of fixed and polymorphic variants in a genetically diverse set of human NANOG and NANOGP8 sequences, we estimated the evolutionary origin of NANOGP8 at approximately 0.9 to 2.5 million years ago, more recent than previously estimated. We also discovered that NANOGP8 arose from a derived variant allele of NANOG containing a 22-nucleotide pair deletion in the 3' UTR, which has remained polymorphic in modern humans. Evidence from our experiments indicates that NANOGP8 is fixed in modern humans even though its parent allele is polymorphic. The presence of NANOGP8-specific sequences in Neanderthal reads provided definitive evidence that NANOGP8 is also present in the Neanderthal genome. Some variants between the reference sequences of NANOG and NANOGP8 utilized in cancer research to distinguish RT-PCR products are polymorphic within NANOG or NANOGP8 and thus are not universally reliable as distinguishing features. NANOGP8 was inserted in reverse orientation into the LTR region of an SVA retroelement that arose in a human-chimpanzee-gorilla common ancestor after divergence of the orangutan ancestral lineage. Transcription factor binding sites within and beyond this LTR may promote expression of NANOGP8 in cancer cells, although current evidence is inferential. The fact that NANOGP8 is a human-specific retro-oncogene may partially explain the higher genetic predisposition for cancer in humans compared with other primates.

Pleiotropic and age-dependent effects of decreased protein modification by O-linked N-acetylglucosamine on pancreatic ß-cell function and vascularization.

The hexosamine biosynthesis pathway (HBP) regulates the post-translational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine (O-GlcNAc). Numerous studies have demonstrated increased flux through this pathway contributes to the development of ß-cell dysfunction. The effect of decreased O-GlcNAc on the maintenance of normal ß-cell function, however, is not well understood. We studied transgenic mice that over express ß-N-acetylglucosaminidase (O-GlcNAcase), an enzyme that catalyzes the removal of O-GlcNAc from proteins, in the pancreatic ß-cell under control of the rat insulin promoter. 3-4-Month-old O-GlcNAcase transgenic mice have higher glucose excursions with a concomitant decrease in circulating insulin levels, insulin mRNA levels, and total islet insulin content. In older (8-9-month-old) O-GlcNAcase transgenic mice glucose tolerance is no longer impaired. This is associated with increased serum insulin, islet insulin content, and insulin mRNA in the O-GlcNAcase transgenic mice. These improvements in ß-cell function with aging are associated with increased angiogenesis and increased VEGF expression, with parallel increases in activation of Akt and expression of PGC1α. The biphasic effects as a function of age are consistent with published observations of mice with increased O-GlcNAc in islets and demonstrate that O-GlcNAc signaling exerts multiple effects on both insulin secretion and islet survival.

Analysis of hepatic glycogen-associated proteins.

Glycogen particles are associated with a population of proteins that mediate its biological functions, including: management of glucose flux into and out of the glycogen particle, maintenance of glycogen structure and regulation of particle size, number, and cellular location. A survey of the glycogen-associated proteome would be predicted to identify the relative representation of known members of this population, and associations with unexpected proteins that have the potential to mediate other functions of the glycogen particle. We therefore purified glycogen particles from both mouse and rat liver, using different techniques, and analyzed the resulting tryptic peptides by MS. We also specifically eluted glycogen-binding proteins from the pellet using malto-oligosaccharides. Comparison of the rat and mouse populations, and analysis of specifically eluted proteins allow some conclusions to be made about the hepatic glycogen sub-proteome. With the exception of glycogen branching enzyme all glycogen metabolic proteins were detected. Novel associations were identified, including ferritin and starch-binding domain protein 1, a protein that contains both a transmembrane endoplasmic reticulum signal peptide and a carbohydrate-binding module. This study therefore provides insight into the organization of the glycogen proteome, identifies other associated proteins and provides a starting point to explore the dynamic nature and cellular distribution of this metabolically important protein population.

Comparative structural analyses of purified glycogen particles from rat liver, human skeletal muscle and commercial preparations.

Glycogen is a cellular energy store that is crucial for whole body energy metabolism, metabolic regulation and exercise performance. To understand glycogen structure we have purified glycogen particles from rat liver and human skeletal muscle tissues and compared their biophysical properties with those found in commercial glycogen preparations. Ultrastructural analysis of commercial liver glycogens fails to reveal the classical alpha-rosette structure but small irregularly shaped particles. In contrast, commercial slipper limpet glycogen consists of beta-particles with similar branching and chain lengths to purified rat liver glycogen together with a tendency to form small alpha-particles, and suggest it should be used as a source of glycogen for all future studies requiring a substitute for mammalian liver glycogen.