Unwin Richard D, Smith Duncan L, Blinco David, Wilson Claire L, Miller Crispin J, Evans Caroline A, Jaworska Ewa, Baldwin Stephen A, Barnes Kay, Pierce Andrew, Spooncer Elaine, Whetton Anthony D
Stem Cell and Leukaemia Proteomics Laboratory, Faculty of Medical and Human Sciences, University of Manchester, Manchester M20 4QL, UK.
Blood. 2006 Jun 15;107(12):4687-94. doi: 10.1182/blood-2005-12-4995. Epub 2006 Feb 28.
The proteome is determined by rates of transcription, translation, and protein turnover. Definition of stem cell populations therefore requires a stem cell proteome signature. However, the limit to the number of primary cells available has restricted extensive proteomic analysis. We present a mass spectrometric method using an isobaric covalent modification of peptides for relative quantification (iTRAQ), which was employed to compare the proteomes of approximately 1 million long-term reconstituting hematopoietic stem cells (Lin(-)Sca(+)Kit(+); LSK(+)) and non-long-term reconstituting progenitor cells (Lin(-)Sca(+)Kit(-); LSK(-)), respectively. Extensive 2-dimensional liquid chromatography (LC) peptide separation prior to mass spectrometry (MS) enabled enhanced proteome coverage with relative quantification of 948 proteins. Of the 145 changes in the proteome, 54% were not seen in the transcriptome. Hypoxia-related changes in proteins controlling metabolism and oxidative protection were observed, indicating that LSK(+) cells are adapted for anaerobic environments. This approach can define proteomic changes in primary samples, thereby characterizing the molecular signature of stem cells and their progeny.
蛋白质组由转录、翻译和蛋白质周转速率决定。因此，干细胞群体的定义需要干细胞蛋白质组特征。然而，可用原代细胞数量的限制阻碍了广泛的蛋白质组学分析。我们提出了一种使用肽的等压共价修饰进行相对定量的质谱方法（iTRAQ），该方法用于分别比较约100万个长期重建造血干细胞（Lin(-)Sca(+)Kit(+); LSK(+)）和非长期重建祖细胞（Lin(-)Sca(+)Kit(-); LSK(-)）的蛋白质组。在质谱分析（MS）之前进行广泛的二维液相色谱（LC）肽分离，可实现增强的蛋白质组覆盖，并对948种蛋白质进行相对定量。在蛋白质组的145种变化中，54%在转录组中未观察到。观察到控制代谢和氧化保护的蛋白质中与缺氧相关的变化，表明LSK(+)细胞适应厌氧环境。这种方法可以定义原代样品中的蛋白质组变化，从而表征干细胞及其后代的分子特征。
