The application of proteomic technologies to the detection of the abuse of gene therapy and protein therapeutic agents

An acetonitrile based protein extraction method was developed that demonstrated high efficient and effective removal of high abundant proteins from both human and murine serum. The protein content of the extract was characterised using gel electrophoresis, the Bradford assay and liquid chromatography tandem mass spectrometry (LC-MS/MS) with database searching. Selected reaction monitoring (SRM) analysis was used to quantify the levels of high abundant serum proteins to further validate the extraction methodology. The ACN depletion method, in combination with artificial neural networks (ANNs) data mining software, was applied to a murine growth hormone (GH) gene doping study with the aim of identifying biomarker ions capable of detecting gene doping. The LC-MS and ANNs analysis approach failed to conclusively identify a biomarker to gene doping in the mouse model. However, the application of the same technique to serum from a rhGH administration study in humans, returned models capable of discriminating between rhGH treated placebo states. The ion identified as being the most discriminatory was characterised using mass spectrometry, and was derived from the protein leucine-rich a-2-glycoprotein (LRG). Multiple LRG related tryptic peptides were identified as being up-regulated upon dosing with recombinant human GH (rhGH). A high throughput LC-MS/MS and SRM approach was developed to quantify proteins in human serum. The approach was validated by comparison of LC-MS/MS derived APO A1 concentrations with those obtained using established clinical analyser technologies. The LC-MS/MS methodology was applied to a large cohort of 257 serum samples from two rhGH administration studies performed at Royal Free Hospital . The two administrations included serum samples from 15 individuals who had been dosed daily with rhGH. Serum concentrations of the established rhGH biomarker insulin-like growth factor-I (IGF-I) were quantified by LC-MS/MS and compared well with those determined using two different immunoassay-based methodologies. Serum concentrations of the LRG protein were measured simultaneously with IGF-I and appeared to increase in 14 of the 15 rhGH dosed individuals. Combining the LRG and IGF-I data further increased the separation of rhGH treated and placebo states within each individual, and the application of ANNs analysis showed that the combination of the two proteins increased the discrimination characteristics over using IGF-I alone. The murine equivalent of the LRG protein was identified and SRM transitions for a tryptically derived peptide were developed, along with transitions for monitoring a peptide from the murine IGF-I protein. These transitions were used to quantify the two proteins in the remaining aliquots from a murine GH gene doping experiment, however neither protein appeared to increase in the GH +ve plasmid samples that were analysed.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Mass spectrometric characterisation of the circulating peptidome following oral glucose ingestion in control and gastrectomised patients.

RATIONALE: Meal ingestion triggers secretion of a variety of gut and endocrine peptides important in diabetes research which are routinely measured by immunoassays. However, similarities between some peptides (glucagon, oxyntomodulin and glicentin) can cause specificity issues with immunoassays. We used a liquid chromatography/tandem mass spectrometry (LC/MS/MS) methodology to unambiguously monitor multiple gut peptides in human plasma. METHODS: A simple acetonitrile-based protein precipitation step, followed by evaporation and solid-phase extraction, removed high-abundance proteins from samples prior to nano-LC/MS/MS analysis on an Orbitrap Q-Exactive Plus mass spectrometer using a data-dependent methodology. Database searching using PEAKS identified multiple gut-derived peptides, including peptides in the mid-pg/mL range. The relative levels of these and previously characterised peptides were assessed in plasma samples from gastrectomised and control subjects during an oral glucose tolerance test. RESULTS: Analysis of plasma extracts revealed significantly elevated levels of a number of peptides following glucose ingestion in subjects who had undergone gastrectomy compared with controls. These included GLP-1(7-36), GLP-1(9-36), glicentin, oxyntomodulin, GIP(1-42), GIP(3-42), PYY(1-36), PYY(3-36), neurotensin, insulin and C-peptide. Motilin levels decreased following glucose ingestion. Results showed good correlation with immunoassay-derived concentrations of some peptides in the same samples. The gastrectomy group also had higher, but non-glucose-dependent, circulating levels of peptides from PIGR and DMBT1. CONCLUSIONS: Overall, the approach showed that a fast, generic and reproducible LC/MS/MS methodology requiring only a small volume of plasma was capable of the multiplexed detection of a variety of diabetes-related peptides.Wellcome Trust and MR

Lesser Snow Geese, Chen caerulescens caerulescens, and Ross's Geese, Chen rossii, of Jenny Lind Island, Nunavut

We surveyed the Lesser Snow (Chen caerulescens caerulescens) and Ross’s geese (Chen rossii) of Jenny Lind Island, Nunavut, using aerial photography in June 1988, 1998, and 2006, and a visual helicopter transect survey in July 1990. The estimated number of nesting geese was 39 154 ± SE 2238 in 1988, 19 253 ± 2323 in 1998, and 21 572 ± 1898 in 2006. In 1988 an estimated 2.7% of the nesting geese were Ross’s. The July 1990 population of adult-plumaged birds was 25 020 ± 3114. The estimated percentage blue morph among Snow and Ross’s geese was 19.0% in 1988, 25.1% in 1989, 23.0% in 1990 and 21.1% in 2006. Estimated pre-fledged Snow Goose productivity was 47% young in 1989 and 46% in 1990. Combined numbers of Snow and Ross’s geese on Jenny Lind Island grew over 250 fold, from 210 adults in 1962-1966 to 54 100 adults in 1985. Numbers subsequently declined, to 42 200 in 1988, 25 000 in 1990, 20 300 in 1998, and 26 400 in 2006. Population decline between 1985 and 1990 was consistent with anecdotal reports by others that die-offs of Snow Geese occurred in 1984, 1985 and 1989, and with our August 1989 fieldwork which found evidence of habitat degradation and malnourishment of young geese. In spite of limited food resources on Jenny Lind Island, the colony continued to exist in 2006 at near its 1990 and 1998 levels. Further studies there could provide insights for management of the overabundant mid-continent Snow Goose population and its arctic habitats

The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer

We have identified a human homolog of the bacterial MutS and S. cerevisiae MSH proteins, called hMSH2. Expression of hMSH2 in E. coli causes a dominant mutator phenotype, suggesting that hMSH2, like other divergent MutS homologs, interferes with the normal bacterial mismatch repair pathway. hMSH2 maps to human chromosome 2p22-21 near a locus implicated in hereditary nonpolyposis colon cancer (HNPCC). A T to C transition mutation has been detected in the -6 position of a splice acceptor site in sporadic colon tumors and in affected individuals of two small HNPCC kindreds. These data and reports indicating that S. cerevisiae msh2 mutations cause an instability of dinucleotide repeats like those associated with HNPCC suggest that hMSH2 is the HNPCC gene

Identification of plasma protease derived metabolites of glucagon and their formation under typical laboratory sample handling conditions

Copyright © 2014 John Wiley & Sons, Ltd. RATIONALE Glucagon modulates glucose production, and it is also a biomarker for several pathologies. It is known to be unstable in human plasma, and consequently stabilisers are often added to samples, although these are not particularly effective. Despite this, there have not been any studies to identify in vitro plasma protease derived metabolites; such a study is described here. Knowledge of metabolism should allow the development of more effective sample stabilisation strategies. METHODS Several novel metabolites resulting from the incubation of glucagon in human plasma were identified using high-resolution mass spectrometry with positive electrospray ionisation. Tandem mass spectrometric (MS/MS) scans were acquired for additional confirmation using a QTRAP. Separation was performed using reversed-phase ultra-high-performance liquid chromatography. The formation of these metabolites was investigated during a time-course experiment and under specific stress conditions representative of typical laboratory handling conditions. Clinical samples were also screened for metabolites. RESULTS Glucagon 3-29 and [pGlu] 3 glucagon 3-29 were the major metabolites detected, both of which were also present in clinical samples. We also identified two oxidised forms of [pGlu] 3 glucagon 3-29 as well as glucagon 19-29 , or 'miniglucagon', along with the novel metabolites glucagon 20-29 and glucagon 21-29 . The relative levels of these metabolites varied throughout the time-course experiment, and under the application of the different sample handling conditions. Aprotinin stabilisation of samples had negligible effect on metabolite formation. CONCLUSIONS Novel plasma protease metabolites of glucagon have been confirmed, and their formation characterised over a time-course experiment and under typical laboratory handling conditions. These metabolites could be monitored to assess the effectiveness of new sample stabilisation strategies, and further investigations into their formation could suggest specific enzyme inhibitors to use to increase sample stability. In addition the potential of the metabolites to affect immunochemistry-based assays as a result of cross-reactivity could be investigated

The in vitro loss of penicillamine in plasma, albumin solutions, and whole blood: Implications for pharmacokinetic studies of penicillamine

The recent development of a high performance liquid chromatography assay method for the analysis of penicillamine in biological samples such as plasma, whole blood, and urine has provided a specific and sensitive assay method to aid in the study of penicillamine pharmacokinetics. Several investigators have reported measuring the plasma concentration of penicillamine. Some of these investigators have indicated that the plasma must be assayed immediately. However, such restrictions can limit the feasibility of a pharmacokinetic study. The results of this paper demonstrate the instability of penicillamine in plasma, albumin solutions, and whole blood. The rate of loss of penicillamine was shown to be influenced by the concentration of albumin. As a result of the significant loss of penicillamine over a short period of time, plasma or whole blood samples must be deproteinated immediately upon collection to avoid the loss of reduced penicillamine. Methods are presented for the preparation of biological samples so that the oxidation of penicillamine is prevented and the samples can be held for several days prior to analysis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23196/1/0000123.pd

Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages

This is the publisher's version. Copyright 2011 by The American Society for Cell Biology.To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP2), several investigators have proposed that there are separate pools of PIP2 in the plasma membrane. Recent experiments show the surface concentration of PIP2 is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP2 are also concentrated in these regions. However, how is the PIP2 produced by these kinases prevented from diffusing rapidly away? First, proteins could act as “fences” around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP2 within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP2. FCS measurements show that PIP2 diffuses rapidly (D ∼ 1 μm2/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP2 does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (∼10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane–anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP2 into and out of forming phagosomes

EAGLE-like simulation models do not solve the entropy core problem in groups and clusters of galaxies

Recent high-resolution cosmological hydrodynamic simulations run with a variety of codes systematically predict large amounts of entropy in the intra-cluster medium at low redshift, leading to flat entropy profiles and a suppressed cool-core population. This prediction is at odds with X-ray observations of groups and clusters. We use a new implementation of the EAGLE galaxy formation model to investigate the sensitivity of the central entropy and the shape of the profiles to changes in the sub-grid model applied to a suite of zoom-in cosmological simulations of a group of mass M500 = 8.8 × 1012 M⊙ and a cluster of mass 2.9 × 1014 M⊙. Using our reference model, calibrated to match the stellar mass function of field galaxies, we confirm that our simulated groups and clusters contain hot gas with too high entropy in their cores. Additional simulations run without artificial conduction, metal cooling or active galactic nuclei (AGN) feedback produce lower entropy levels but still fail to reproduce observed profiles. Conversely, the two objects run without supernova feedback show a significant entropy increase which can be attributed to excessive cooling and star formation. Varying the AGN heating temperature does not greatly affect the profile shape, but only the overall normalization. Finally, we compared runs with four AGN heating schemes and obtained similar profiles, with the exception of bipolar AGN heating, which produces a higher and more uniform entropy distribution. Our study leaves open the question of whether the entropy core problem in simulations, and particularly the lack of power-law cool-core profiles, arise from incorrect physical assumptions, missing physical processes, or insufficient numerical resolution

The Human and Mouse Islet Peptidome: Effects of Obesity and Type 2 Diabetes, and Assessment of Intraislet Production of Glucagon-like Peptide-1.

To characterize the impact of metabolic disease on the peptidome of human and mouse pancreatic islets, LC-MS was used to analyze extracts of human and mouse islets, purified mouse alpha, beta, and delta cells, supernatants from mouse islet incubations, and plasma from patients with type 2 diabetes. Islets were obtained from healthy and type 2 diabetic human donors, and mice on chow or high fat diet. All major islet hormones were detected in lysed islets as well as numerous peptides from vesicular proteins including granins and processing enzymes. Glucose-dependent insulinotropic peptide (GIP) was not detectable. High fat diet modestly increased islet content of proinsulin-derived peptides in mice. Human diabetic islets contained increased content of proglucagon-derived peptides at the expense of insulin, but no evident prohormone processing defects. Diabetic plasma, however, contained increased ratios of proinsulin and des-31,32-proinsulin to insulin. Active GLP-1 was detectable in human and mouse islets but 100-1000-fold less abundant than glucagon. LC-MS offers advantages over antibody-based approaches for identifying exact peptide sequences, and revealed a shift toward islet insulin production in high fat fed mice, and toward proglucagon production in type 2 diabetes, with no evidence of systematic defective prohormone processing