SGER: Proteomics as a Tool for Cloning Leptin from Fence Lizard Sceloperus Undulatus

SGER:蛋白质组学作为克隆篱笆蜥蜴瘦素的工具

基本信息

  • 批准号:
    0328554
  • 负责人:
  • 金额:
    $ 8万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
    Standard Grant
  • 财政年份:
    2003
  • 资助国家:
    美国
  • 起止时间:
    2003-08-01 至 2005-07-31
  • 项目状态:
    已结题

项目摘要

Proteomics as a tool for Cloning Leptin from fence lizard Sceloporus undulatusPeter H. Niewiarowski1, Richard L. Londraville1, Michael Kinter21The University of Akron and 2The Cleveland Clinic FoundationFat metabolism centrally influences physiological choices that organisms make with the calories they acquire. Whether the goal is to discover the molecular basis of obesity in humans, or to understand how and when a hibernator accumulates fat to survive the winter, studying proteins that influence fat metabolism gives insight into those processes. Leptin is a recently discovered protein hormone that affects both the anabolic (fat storage) and catabolic (fat burning) aspects of fat metabolism. Because leptin has the potential to explain so many aspects of fat metabolism, it has been studied intensely (6000 studies since 1994). Nearly all of these studies have been focused on mammals, with relatively little attention paid to ectotherms. Studying leptin (and other proteins that influence fat metabolism) in non-mammals brings the historically strong approach of comparative biology to studies of fat metabolism. Although leptin has been cloned and sequenced in dozens of mammals, no sequence is published for any ectotherms despite considerable effort. This award funds a new approach, proteomics, to cloning and characterizing leptin from fence lizard (Sceloporus undulatus). Proteomics is the study of all proteins present in a given tissue at a given time. Under previous funding (IBN-0099303 to PHN and RLL), a protein in lizard blood and brain that binds to leptin antibodies and is the same size as mammalian leptins was identified. Concentrations of this putative lizard leptin were measured in natural populations of fence lizards; lizard leptin peaks in the spring and summer and then declines significantly in the fall as the lizards accumulate fat and prepare for winter hibernation. This is consistent with the pattern of leptin concentrations described for some mammalian hibernators. It is possible now to use this pattern of seasonal variation to compare the proteomes of lizards at their peak and nadir of leptin concentrations. Proteins from liver, brain, fat bodies, and serum will be extracted and separated according to their charge (first dimension) and size (second dimension) by gel electrophoresis. This procedure results in a two-dimensional 'map' of proteins that is characteristic for each tissue. The two-dimensional maps will be scanned and compared (via digital imaging) between fall and summer lizards from the same population. All proteins that change in relative amount with season will be submitted to mass-spectrometry analyses for identification. The mass-spectrometry experiments can determine the identity of the protein by cutting it at known amino acids (protein digest) and then measuring the mass of the resulting pieces; this collection of masses is compared to all known protein sequences, all of which have been cut the same way (virtually, by a computer). If there is no match, the unknown can be further fractionated to determine its amino acid sequence. This amino acid sequence can subsequently be used as the basis for traditional molecular cloning experiments and other experiments to determine the function of the novel protein.The broader impacts of the proposed research are that it will promote teaching, training, and learning via incorporation of proteomics into laboratory exercises for undergraduates at the University of Akron (UA). PHN and RLL both hold current teaching scholarship grants (GK-12 and CCLI) in which integration of research and teaching is an essential goal. Therefore, this research will be used seamlessly to enhance learning for undergraduate and graduate students. The proposal will also broaden the participation of underrepresented groups. UA Biology has a significant minority student population, and they will be exposed to the proposed research. In addition, PN and RL have successfully in the past, and will continue to recruit underrepresented groups to work on the proposed research. By establishing a new partnership between UA and The Cleveland Clinic Foundation, we will enhance infrastructure for research and education. The benefits of the proposed activity to society are significant. This research is a new approach to studying a hormone (leptin) that is established to play a role in the global health epidemic of obesity.
蛋白质组学作为从栅栏蜥蜴Scelopus undulatusPeter H.Niewiarowski 1,Richard L.Londraville1,Michael Kinter21克隆瘦素的工具,阿克伦大学和2克利夫兰临床基金会脂肪代谢集中影响生物体利用他们获得的卡路里做出的生理选择。无论目标是发现人类肥胖的分子基础,还是了解冬眠者如何以及何时积累脂肪以度过冬天,研究影响脂肪新陈代谢的蛋白质都能深入了解这些过程。瘦素是最近发现的一种蛋白质激素,它同时影响脂肪代谢的合成代谢(脂肪储存)和分解代谢(脂肪燃烧)方面。由于瘦素有可能解释脂肪代谢的许多方面,人们对其进行了深入的研究(自1994年以来共进行了6000项研究)。几乎所有这些研究都集中在哺乳动物身上,相对较少关注外温类动物。在非哺乳动物中研究瘦素(和其他影响脂肪代谢的蛋白质)为脂肪代谢研究带来了历史上强大的比较生物学方法。尽管瘦素已经在数十种哺乳动物身上被克隆和测序,但尽管付出了相当大的努力,但还没有公布任何外胚层动物的序列。该奖项资助了一种新的方法,蛋白质组学,以克隆和表征栅栏蜥蜴(Scelopus Undulatus)的瘦素。蛋白质组学是研究在给定时间存在于给定组织中的所有蛋白质。在之前的资助下(从IBN-0099303到PHN和RLL),在蜥蜴血和脑中发现了一种与瘦素抗体结合的蛋白质,其大小与哺乳动物的瘦素相同。在栅栏蜥蜴的自然种群中测量了这种假定的蜥蜴瘦素的浓度;蜥蜴瘦素在春季和夏季达到峰值,然后在秋季显着下降,因为蜥蜴积累脂肪,为冬季冬眠做准备。这与一些哺乳动物冬眠者的瘦素浓度模式是一致的。现在有可能利用这种季节性变化模式来比较蜥蜴瘦素浓度峰值和最低点的蛋白质组。从肝脏、脑、脂肪体和血清中提取蛋白质,并根据其电荷(第一维)和大小(第二维)进行凝胶电泳法分离。这一过程产生了一张具有每个组织特征的蛋白质的二维“图谱”。这些二维地图将被扫描,并(通过数字成像)在同一种群的秋季和夏季蜥蜴之间进行比较。所有相对含量随季节变化的蛋白质将被提交给质谱分析进行鉴定。质谱学实验可以通过在已知氨基酸(蛋白质消化)处切割蛋白质,然后测量得到的片段的质量来确定蛋白质的身份;将这些质量块与所有已知的蛋白质序列进行比较,所有这些序列都以相同的方式(虚拟地,由计算机)切割。如果没有匹配,则可以进一步分离未知基因以确定其氨基酸序列。这一氨基酸序列随后可以作为传统的分子克隆实验和其他实验的基础,以确定这种新蛋白质的功能。拟议的研究的更广泛影响是,它将通过将蛋白质组学纳入阿克伦大学(UA)本科生的实验室练习来促进教学、培训和学习。PHN和RLL都拥有目前的教学奖学金(GK-12和CCLI),其中研究和教学一体化是一个基本目标。因此,这项研究将被无缝地用于促进本科生和研究生的学习。该提案还将扩大代表性不足群体的参与范围。UA Biology拥有相当大的少数族裔学生群体,他们将接触到拟议的研究。此外,PN和RL在过去取得了成功,并将继续招募代表人数不足的团体参与拟议的研究。通过在UA和克利夫兰临床基金会之间建立新的合作伙伴关系,我们将加强研究和教育的基础设施。拟议的活动对社会的好处是显著的。这项研究是研究一种激素(瘦素)的新方法,瘦素被确立为在全球肥胖症的健康流行中发挥作用。

项目成果

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Peter Niewiarowski其他文献

Peter Niewiarowski的其他文献

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{{ truncateString('Peter Niewiarowski', 18)}}的其他基金

Workshop: Designing a Network for Undergraduate Biomimicry Research and Education; October 6-8, 2017, Cleveland/Akron, Ohio
研讨会:设计本科生仿生学研究和教育网络;
  • 批准号:
    1747598
  • 财政年份:
    2017
  • 资助金额:
    $ 8万
  • 项目类别:
    Standard Grant
GK-12 Formal Proposal
GK-12 正式提案
  • 批准号:
    0086378
  • 财政年份:
    2001
  • 资助金额:
    $ 8万
  • 项目类别:
    Continuing Grant
Isolating and Characterizing the Fat-Regulating Hormone Leptin in the Fence Lizard
栅栏蜥蜴中脂肪调节激素瘦素的分离和表征
  • 批准号:
    0099303
  • 财政年份:
    2001
  • 资助金额:
    $ 8万
  • 项目类别:
    Standard Grant

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    nhmrc : 1056147
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Valens: A Mass Spectrometry-Based Antibody Sequencing Tool by Digital Proteomics
Valens:数字蛋白质组学基于质谱的抗体测序工具
  • 批准号:
    8474794
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Valens: A Mass Spectrometry-Based Antibody Sequencing Tool by Digital Proteomics
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    8311604
  • 财政年份:
    2012
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"Microplate reader, a necessary tool for functional genomics and proteomics studies in animal, microbial and plant systems"
“酶标仪,动物、微生物和植物系统功能基因组学和蛋白质组学研究的必要工具”
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    422258-2012
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    2011
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PATTERNLAB FOR PROTEOMICS: A TOOL FOR DIFFERENTIAL SHOTGUN PROTEOMICS
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EAGER: Tool development for proteomics and environmental metaproteomics
EAGER:蛋白质组学和环境宏蛋白质组学工具开发
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利用蛋白质组学工具综合分析自身免疫性肝炎中的自身抗体
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  • 批准号:
    7957737
  • 财政年份:
    2009
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    $ 8万
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A QUANTITATIVE ANALYSIS SOFTWARE TOOL FOR MASS SPECTROMETRY-BASED PROTEOMICS
基于质谱的蛋白质组学定量分析软件工具
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X-tracker: a generic quantitation tool for MS-based proteomics:
X-tracker:基于 MS 的蛋白质组学通用定量工具:
  • 批准号:
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