Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry
Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry
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DOI：
10.1038/nbt1001-946
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发表时间：
2001-10-01
影响因子：
46.9
通讯作者：
Aebersold, R
中科院分区：
文献类型：
--
作者：
Han, DK;Eng, J;Aebersold, R
本文描述了一种系统鉴定和定量细胞微粒体组分中所含蛋白质的方法。它包括三个步骤：（1）从代表不同状态的细胞或组织中制备微粒体级分;（2）用同位素编码的亲和标签（ICAT）试剂共价标记蛋白质，然后对组合的标记蛋白质样品进行蛋白水解;和（3）通过多维色谱法、自动串联质谱法和所获得数据的计算分析来分离、鉴定和定量标记的肽。该方法被用来识别和确定的491个蛋白质中的每一个的丰度比的微粒体组分的幼稚和体外分化的人髓性白血病（HL-60）细胞。该方法和支持它的新软件工具非常适合于膜蛋白和标准蛋白质组学技术难以处理的其他类型蛋白质的大规模定量分析。
An approach to the systematic identification and quantification of the proteins contained in the microsomal fraction of cells is described. It consists of three steps: (1) preparation of microsomal fractions from cells or tissues representing different states; (2) covalent tagging of the proteins with isotope-coded affinity tag (ICAT) reagents followed by proteolysis of the combined labeled protein samples; and (3) isolation, identification, and quantification of the tagged peptides by multidimensional chromatography, automated tandem mass spectrometry, and computational analysis of the obtained data. The method was used to identify and determine the ratios of abundance of each of 491 proteins contained in the microsomal fractions of naive and in vitro-differentiated human myeloid leukemia (HL-60) cells. The method and the new software tools to support it are well suited to the large-scale, quantitative analysis of membrane proteins and other classes of proteins that have been refractory to standard proteomics technology.