Mapping intact protein isoforms in discovery mode using top-down proteomics.
Mapping intact protein isoforms in discovery mode using top-down proteomics.
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DOI：
10.1038/nature10575
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发表时间：
2011-10-30
期刊：
影响因子：
64.8
通讯作者：
Kelleher, Neil L.
中科院分区：
文献类型：
--
作者：
Tran, John C.;Zamdborg, Leonid;Ahlf, Dorothy R.;Lee, Ji Eun;Catherman, Adam D.;Durbin, Kenneth R.;Tipton, Jeremiah D.;Vellaichamy, Adaikkalam;Kellie, John F.;Li, Mingxi;Wu, Cong;Sweet, Steve M. M.;Early, Bryan P.;Siuti, Nertila;LeDuc, Richard D.;Compton, Philip D.;Thomas, Paul M.;Kelleher, Neil L.
人类蛋白质组的完整描述依赖于检测体内成熟和变化形式的蛋白质分子的挑战性任务。大规模蛋白质组分析通常涉及消化完整蛋白质，然后使用质谱法（MS）进行推断蛋白质鉴定。这种“自下而上”的过程提供了大量的鉴定（并不总是对单个基因唯一的）。然而，复杂性来自于可变剪接形式的不完整或模糊的表征，不同的修饰（例如，乙酰化和甲基化）和内源性蛋白质裂解，特别是当这些的组合产生完整蛋白质同种型和种类的复杂模式时。对整个蛋白质的“自上而下”的询问可以克服单个蛋白质的这些问题，但是由于缺乏与串联MS很好地整合的完整蛋白质分级分离方法，在蛋白质组规模上还没有实现。在这里，我们显示，使用新的四维（4D）分离系统，鉴定了来自人类细胞的1，043个基因产物，这些基因产物分散在>3，通过翻译后修饰、RNA剪接和蛋白水解产生的000种蛋白质。整个系统在分离能力和蛋白质组覆盖率方面都产生了>20倍的增加，使得能够鉴定高达105千道尔顿的蛋白质和具有高达11个跨膜螺旋的蛋白质。许多以前未检测到的内源性人类蛋白质的异构体被映射，包括响应于DNA损伤诱导的加速细胞老化（衰老）的多重修饰物种的变化。与最新版本的Swiss-Prot数据库相结合，这些数据提供了与单个基因的精确相关性，并为整个蛋白质分子的大规模询问提供了概念验证。该技术有望改善基础生物学和疾病研究中蛋白质组学数据与复杂表型之间的联系。
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 (MS). 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 (e.g., 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 due to the lack of intact protein fractionation methods that are well integrated with tandem MS. Here we show, using a new four dimensional (4D) separation system, identification of 1,043 gene products from human cells that are dispersed into >3,000 protein species created by post-translational modification, RNA splicing, and proteolysis. The overall system produced >20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kilodaltons and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply-modified species in response to accelerated cellular aging (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.
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影响因子：
7
作者：
Patrie, SM;Ferguson, JT;Kelleher, NL
通讯作者：
Kelleher, NL
DOI：
10.1016/j.jasms.2009.08.001
发表时间：
2009-12
影响因子：
3.2
作者：
Lee JE;Kellie JF;Tran JC;Tipton JD;Catherman AD;Thomas HM;Ahlf DR;Durbin KR;Vellaichamy A;Ntai I;Marshall AG;Kelleher NL
通讯作者：
Kelleher NL
DOI：
10.1111/j.2517-6161.1995.tb02031.x
发表时间：
1995-01-01
影响因子：
5.8
作者：
BENJAMINI, Y;HOCHBERG, Y
通讯作者：
HOCHBERG, Y
影响因子：
3.4
作者：
Durbin, Kenneth R.;Tran, John C.;Zamdborg, Leonid;Sweet, Steve M. M.;Catherman, Adam D.;Lee, Ji Eun;Li, Mingxi;Kellie, John F.;Kelleher, Neil L.
通讯作者：
Kelleher, Neil L.
影响因子：
46.9
作者：
Meng, FY;Cargile, BJ;Kelleher, NL
通讯作者：
Kelleher, NL