Ficarro Scott B, Zhang Yi, Lu Yu, Moghimi Ahmadali R, Askenazi Manor, Hyatt Elzbieta, Smith Eric D, Boyer Leah, Schlaeger Thorsten M, Luckey C John, Marto Jarrod A
Department of Cancer Biology, Dana-Farber Cancer Institute, 44 Binney Street, Smith 1158A, Boston, Massachusetts 02115-6084, USA.
Anal Chem. 2009 May 1;81(9):3440-7. doi: 10.1021/ac802720e.
Characterization of signaling pathways in embryonic stem cells is a prerequisite for future application of these cells to treat human disease and other disorders. Identification of tyrosine signaling cascades is of particular interest but is complicated by the relatively low levels of tyrosine phosphorylation in embryonic stem cells. These hurdles correlate with the primary limitations of mass spectrometry-based proteomics; namely, poor detection limit and dynamic range. To overcome these obstacles, we fabricated miniaturized LC-electrospray assemblies that provided approximately 15-fold improvement in LC-MS performance. Significantly, our characterization data demonstrate that electrospray ionization efficiency compensates for diminished chromatographic performance at effluent flow rates below Van Deemter minima. Use of these assemblies facilitated quantitative proteomics-based analysis of tyrosine signaling cascades in embryonic stem cells. Our results suggest that a renewed focus on miniaturized LC coupled to ultralow flow electrospray will provide a viable path for proteomic analysis of primary cells and rare post-translational modifications.
胚胎干细胞中信号通路的表征是未来将这些细胞应用于治疗人类疾病和其他病症的先决条件。酪氨酸信号级联的鉴定尤为重要，但由于胚胎干细胞中酪氨酸磷酸化水平相对较低而变得复杂。这些障碍与基于质谱的蛋白质组学的主要局限性相关；即检测限和动态范围较差。为了克服这些障碍，我们制造了小型化的液相色谱 - 电喷雾组件，其在液相色谱 - 质谱性能方面提供了约15倍的提升。值得注意的是，我们的表征数据表明，在流出物流量低于范德姆特最小值时，电喷雾电离效率可补偿色谱性能的下降。使用这些组件有助于基于定量蛋白质组学分析胚胎干细胞中的酪氨酸信号级联。我们的结果表明，重新关注与超低流量电喷雾耦合的小型化液相色谱将为原代细胞和罕见翻译后修饰的蛋白质组学分析提供一条可行的途径。
