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.

对于按分子量 (MW) 对完整蛋白质进行分级分离，提出了一种大幅改进的二维 (2D) 分离方法，以在复杂混合物的自上而下质谱分析之前推动可重现和稳健的分级分离。“GELFrEE”（即凝胶洗脱液体组分包埋电泳）方法是通过使用 Tris-甘氨酸和 Tris-tricine 凝胶系统实施的，该系统应用于 HeLa S3 细胞的人细胞溶质和核提取物，以实现基于 MW 的分离1 小时内蛋白质从 5 到 >100 kDa。对于低质量蛋白质组 (5–25 kDa) 的自上而下串联质谱 (MS/MS)，5 到 8 个凝胶洗脱 (GE) 组分通过纳米毛细管-LC-MS/MS 以 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 种蛋白质，其中大约一半产生自动 ProSight 识别。介绍了该系统的重现性指标，以及有丝分裂与异步细胞中蛋白质靶标的比较分析。我们转发这种基本的 2D 方法，以促进自上而下质谱法和各种其他蛋白质分离和/或表征方法的更广泛实施。