A proteomic analysis of human cilia - Identification of novel components
A proteomic analysis of human cilia - Identification of novel components
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DOI：
10.1074/mcp.m200037-mcp200
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发表时间：
2002-06-01
影响因子：
7
通讯作者：
Boucher, RC
中科院分区：
文献类型：
--
作者：
Ostrowski, LE;Blackburn, K;Boucher, RC
纤毛在保护呼吸道中起着重要的作用，它为纤毛粘液的清除提供了必要的力量。虽然已经描述了人类纤毛的主要结构成分，但要全面了解纤毛的功能和调节，还需要对所有纤毛成分进行鉴定和表征。根据鞭毛衣单胞菌的研究估计，一个轴突体含有2.250个蛋白质。为了鉴定人类纤毛的所有成分，我们已经开始对分离的纤毛轴突基因组进行全面的蛋白质组学分析。通过二维（2-D） PAGE分析，得到了一个由240多个良好分辨成分组成的高度可重复性的2-D图谱。单个蛋白点用胰蛋白酶消化，用液相色谱/串联质谱（LC/MS/MS）测序。通过该方法获得了38个潜在纤毛蛋白的肽匹配。为了鉴定未被二维PAGE分解的纤毛成分，轴突蛋白在一维凝胶上分离。凝胶通道被分成45个单独的切片，每个切片都被LC/MS/ MS分析，实验结果与另外110个蛋白质的肽匹配。在第三种方法中，分离的轴突组用Lys-C酶切，得到的肽直接通过LC/MS/MS或多维LC/MS/MS进行分析，从而鉴定出另外66种蛋白质。这四种方法中的每一种都能鉴定出存在的蛋白质子集。总共获得了1400多个多肽的序列数据，并鉴定了200多个潜在的轴突蛋白。还获得了200多个人类表达序列标签的肽段匹配。为了验证质谱分析结果，进一步的研究检测了纤毛或纤毛细胞中几种鉴定蛋白（膜联蛋白1、精子蛋白Sp17、视网膜色素变性蛋白RP1）的表达。这些研究代表了人类纤毛轴突的第一个蛋白质组学分析，并确定了这个复杂细胞器的许多潜在的新成分。
Cilia play an essential role in protecting the respiratory tract by providing the force necessary for mucociliary clearance. Although the major structural components of human cilia have been described, a complete understanding of cilia function and regulation will require identification and characterization of all ciliary components. Estimates from studies of Chlamydomonas flagella predict that an axoneme contains 2: 250 proteins. To identify all the components of human cilia, we have begun a comprehensive proteomic analysis of isolated ciliary axon-emes. Analysis by two-dimensional (2-D) PAGE resulted in a highly reproducible 2-D map consisting of over 240 well resolved components. Individual protein spots were digested with trypsin and sequenced using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Peptide matches were obtained to 38 potential ciliary proteins by this approach. To identify ciliary components not resolved by 2-D PAGE, axonemal proteins were separated on a one-dimensional gel. The gel lane was divided into 45 individual slices, each of which was analyzed by LC/MS/ MS. This experiment resulted in peptide matches to an additional 110 proteins. In a third approach, preparations of isolated axonemes were digested with Lys-C, and the resulting peptides were analyzed directly by LC/MS/MS or by multidimensional LC/MS/MS, leading to the identification of a further 66 proteins. Each of the four approaches resulted in the identification of a subset of the proteins present. In total, sequence data were obtained on over 1400 peptides, and over 200 potential axonemal proteins were identified. Peptide matches were also obtained to over 200 human expressed sequence tags. As an approach to validate the mass spectrometry results, additional studies examined the expression of several identified proteins (annexin 1, sperm protein Sp17, retinitis pigmentosa protein RP1) in cilia or ciliated cells. These studies represent the first proteomic analysis of the human ciliary axoneme and have identified many potentially novel components of this complex organelle.