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.

真核细胞的显著特征在于核的存在，它将基因组与细胞质分隔开来。包括转录、DNA复制、RNA加工和核糖体生物合成在内的关键细胞事件均发生在细胞核内。所有这些过程均可通过蛋白质在核膜上的调控性双向转位进行调节，使得细胞核成为一个高度动态的细胞器。在本研究中，我们提出了四种正交的多维分离技术，以全面表征酵母细胞核蛋白质组。通过结合肽水平（SCX色谱、等电聚焦）和蛋白质水平（SDS-PAGE、磷酸纤维素色谱）的方法，并结合质谱分析，我们从高度纯化的细胞核中鉴定出1889种蛋白质，其中1032种此前已被标注为核蛋白。特别是，最成功的方案是使用磷酸纤维素P11色谱结合SDS-PAGE和反相色谱。磷酸纤维素P11色谱传统上用于转录调控中涉及的蛋白质复合物的纯化。在此，通过与LC-MS的结合，该方法比其他三种方法联合使用时鉴定出的蛋白质数量多出约1.5倍，从而显著提高了核蛋白的覆盖率。此外，使用该技术还实现了DNA结合蛋白的富集，并证明是分析多个蛋白质复合物的宝贵工具。对特定核复合物的富集导致了高蛋白质序列覆盖率，这对于亚基的详细表征将尤为有用。