University of Illinois Urbana-Champaign, Department of Chemistry and The Institute for Genomic Biology, Urbana, Illinois 61801, USA.
J Am Soc Mass Spectrom. 2009 Dec;20(12):2183-91. doi: 10.1016/j.jasms.2009.08.001. Epub 2009 Aug 12.
For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The "GELFrEE" (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5-25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.
为了对完整蛋白质进行分子量（MW）的分段，我们提出了一种显著改进的二维（2D）分离方法，以便在复杂混合物的自上而下的质谱分析之前进行可重复且稳健的分段。“GELFrEE”（即凝胶洗脱液相捕集电泳）方法通过使用 Tris-甘氨酸和 Tris-三嗪凝胶系统在 HeLa S3 细胞的人细胞质和核提取物中实施，可在 1 小时内实现 MW 基础上的蛋白质分段，分子量范围为 5 至>100 kDa。对于低分子量蛋白质组（5-25 kDa）的自上而下串联质谱（MS/MS）分析，通过纳米毛细管-LC-MS/MS 对 5 至 8 个凝胶洗脱（GE）馏分进行采样，使用 12 或 14.5 特斯拉傅里叶变换离子回旋共振（FT-ICR）质谱仪。单次注射可检测到约 40 种可检测蛋白质，其中约有一半可通过自动 ProSight 鉴定。本系统的重现性指标以及有丝分裂与非同步细胞中蛋白质靶标的比较分析也一并呈现。我们提出了这种基本的 2D 方法，以促进自上而下的质谱分析以及各种其他蛋白质分离和/或表征方法的广泛应用。
