Orthogonal Separation Techniques for the Characterization of the Yeast Nuclear Proteome

The presence of the nucleus is the distinguishing feature of eukaryotic cells, separating the genome from the cytoplasm. Key cellular events, including transcription, DNA replication, RNA-processing and ribosome biogenesis all take place in the nucleus. All of these processes can be regulated through controlled and bidirectional translocation of proteins across the nuclear envelope, making the nucleus a highly dynamic organelle. In this study, we present four orthogonal multidimensional separation techniques for the comprehensive characterization of the yeast nuclear proteome. By combining methods on the peptide level (SCX chromatography, isoelectric focusing) and protein level (SDS-PAGE, phosphocellulose chromatography) coupled with mass spectrometry, we identified 1889 proteins from highly purified nuclei, of which 1032 were previously annotated as nuclear proteins. In particular, the most successful setup was the use of phosphocellulose P11 chromatography in combination with SDS-PAGE and reversed phase chromatography. Phosphocellulose P11 chromatography has been classically used for the purification of functional protein complexes involved in transcription regulation. Here, by its coupling with LC-MS, this method resulted in approximately 1.5 times more protein identifications than the other three combined, thereby contributing significantly to the coverage of nuclear proteins. In addition, the use of this technique resulted in the enrichment of DNA binding proteins and proved to be a valuable tool for the simultaneous analysis of multiple protein complexes. The enrichment for specific nuclear complexes has resulted in high protein sequence coverage, which will be particularly useful for the detailed characterization of subunits.细胞核的存在是真核细胞的标志性特征，可将基因组与细胞质分隔开来。包括转录、DNA复制、RNA加工以及核糖体生物发生在内的关键细胞事件均发生于细胞核内。上述所有过程均可通过蛋白质跨核被膜的受控双向转运进行调控，这使得细胞核成为一种高度动态的细胞器。本研究采用四种正交多维分离技术，对酵母核蛋白质组进行全面表征。研究团队结合肽水平的强阳离子交换色谱（SCX chromatography）、等电聚焦，与蛋白质水平的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、磷酸纤维素色谱等分离方法，并与质谱联用，从高纯度细胞核中鉴定出1889种蛋白质，其中1032种此前已被注释为核蛋白质。尤为关键的是，表现最优的实验方案是将磷酸纤维素P11色谱与十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）及反相色谱联用。磷酸纤维素P11色谱传统上多用于纯化参与转录调控的功能性蛋白质复合物。本研究中将其与液相色谱-质谱联用（LC-MS）结合后，该方法鉴定得到的蛋白质数量约为其余三种方法总和的1.5倍，极大地提升了核蛋白质的覆盖度。此外，该技术可实现DNA结合蛋白的富集，同时也是同时分析多种蛋白质复合物的有效工具。对特定核复合物的富集实现了较高的蛋白质序列覆盖度，这对于亚基的精细表征具有重要应用价值。