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Mapping intact protein isoforms in discovery mode using top-down proteomics

机译:使用自上而下的蛋白质组学在发现模式下定位完整的蛋白质同工型

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摘要

A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry. This' bottom-up' process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species. 'Top-down' interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmem-brane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research.
机译:对人类蛋白质组的完整描述依赖于检测体内蛋白质分子成熟和变化形式的艰巨任务。大规模蛋白质组分析通常涉及消化完整蛋白,然后使用质谱分析法推断蛋白。这种“自下而上”的过程提供了大量的鉴定(并非总是单个基因唯一)。但是,复杂性是由其他剪接形式的不完整或模棱两可的特性,各种修饰(例如,乙酰化和甲基化)和内源性蛋白裂解引起的,特别是当这些结合使用时会形成完整蛋白同工型和物种的复杂模式时。整个蛋白质的“自上而下”查询可以克服单个蛋白质的这些问题,但是由于缺乏与串联质谱法很好地集成的完整蛋白质分级分离方法,因此在蛋白质组学上尚未实现。在这里,我们显示了使用新的三维分离系统,鉴定了来自人类细胞的1,043个基因产物,这些产物被分散到通过翻译后修饰(PTM),RNA剪接和蛋白水解产生的3,000多种蛋白质中。整个系统的分离能力和蛋白质组覆盖率均提高了20倍以上,从而能够鉴定高达105 kDa的蛋白质和具有11个跨膜螺旋的蛋白质。绘制了许多以前未发现的内源人类蛋白质同工型,包括响应DNA损伤引起的加速细胞衰老(衰老)的多重修饰物种的变化。数据与Swiss-Prot数据库的最新版本集成,可为单个基因提供精确的关联,并为大规模询问整个蛋白质分子提供概念验证。该技术有望改善蛋白质组学数据与基础生物学和疾病研究中复杂表型之间的联系。

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  • 来源
    《Nature》 |2011年第7376期|p.254-258|共5页
  • 作者单位

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Doping Control Center and Center for Theragnosis, Korea Institute of Science and Technology, Seoul, South Korea;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA;

    Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Department of Nanomedicine, The Methodist Hospital Research Institute, Houston, Texas 77030, USA (A.V.) Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA (N.S.);

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Department of Nanomedicine, The Methodist Hospital Research Institute, Houston, Texas 77030, USA (A.V.) Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA (N.S.);

    Department of Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA;

    Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA;

    Departments of Chemistry and Biochemistry, and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Departments of Chemistry and Molecular Biosciences and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60208, USA;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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  • 正文语种 eng
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