An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture
An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture
复制标题
DOI：
10.1074/mcp.m800190-mcp200
复制
发表时间：
2009-03-01
影响因子：
7
通讯作者：
Lajoie, Gilles A.
中科院分区：
文献类型：
--
作者：
Bendall, Sean C.;Hughes, Chris;Lajoie, Gilles A.
人胚胎干细胞（hESC）的衍生和长期维持已经建立在依赖和独立于支持（饲养）细胞的培养形式中。然而，负责保持hESC在新生状态下的活力的因素仍然未知。我们描述了一种基于质谱的方法，用于探测人胚胎干细胞培养微环境的分泌组，以识别低丰度的潜在调节蛋白因子。使用连续质量（m/z）和保留时间定向排除法对单个样品进行多次分析，而不对相同的肽离子进行两次采样。这种迭代排除质谱法（IE-MS）方法与同一仪器上的简单重复分析方法相比，即使在大量样品预分离后，蛋白质和肽指标也增加了一倍以上。此外，IE-MS方法的实施，以提高性能的一个较旧的四极杆飞行时间（Q-ToF）MS。所得到的识别肽的数量接近一个新的LTQ-Orbitrap MS上的平行重复分析。这两种仪器的结果相结合被证明是上级，以实现由一个单一的仪器在识别额外的蛋白质。使用IE-MS策略，结合互补的基于凝胶和溶液的分级分离方法，广泛探索hESC培养微环境。与以前发表的调查相比，观察到超过10至12倍的细胞外蛋白。检测到以前无法检测到的生长因子，浓度范围从10（-9）到10（-11）μ g/ml，突出了我们分析的深度。IE-MS方法提供了一种简单可靠的技术，通过增加基于MS的蛋白质组学分析的有效深度，大大提高了仪器性能。这种方法应广泛适用于任何LC-MS/MS仪器平台或生物系统。Molecular & Cellular Proteomics 8：421-432，2009.
The derivation and long-term maintenance of human embryonic stem cells (hESCs) has been established in culture formats that are both dependent and independent of support (feeder) cells. However, the factors responsible for preserving the viability of hESCs in a nascent state remain unknown. We describe a mass spectrometry-based method for probing the secretome of the hESC culture microenvironment to identify potential regulating protein factors that are in low abundance. Individual samples were analyzed several times, using successive mass (m/z) and retention time-directed exclusion, without sampling the same peptide ion twice. This iterative exclusion-mass spectrometry (IE-MS) approach more than doubled protein and peptide metrics in comparison to a simple repeat analysis method on the same instrument, even after extensive sample pre-fractionation. Furthermore, implementation of the IE-MS approach was shown to enhance the performance of an older quadrupole time of flight (Q-ToF) MS. The resulting number of identified peptides approached that of a parallel repeat analysis on a newer LTQ-Orbitrap MS. The combination of the results of both instruments proved to be superior to that achieved by a single instrument in the identification of additional proteins. Using the IE-MS strategy, combined with complementary gel- and solution-based fractionation methods, the hESC culture microenvironment was extensively probed. Over 10 to 12 times more extracellular proteins were observed compared with previously published surveys. The detection of previously undetectable growth factors, present at concentrations ranging from 10(-9) to 10(-11) g/ml, highlights the depth of our profiling. The IE-MS approach provides a simple and reliable technique that greatly enhances instrument performance by increasing the effective depth of MS-based proteomic profiling. This approach should be widely applicable to any LC-MS/MS instrument platform or biological system. Molecular & Cellular Proteomics 8:421-432, 2009.