Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
J Proteome Res. 2009 Nov;8(11):5020-30. doi: 10.1021/pr900449e.
The basal transcription factor TFIID and the chromatin-modifying complex SAGA, which have several subunits in common, are crucial for transcription regulation. Here, we describe an in-depth profiling of post-translational modifications (PTMs) on both TFIID and SAGA from yeast. We took a multipronged approach using high-resolution mass spectrometry (LC-MS) in combination with the proteases Trypsin, Chymotrypsin and Glu-C. The cumulative peptide identification data, at a false discovery rate <1%, allowed us to cover most TFIID and SAGA subunit sequences to near completion. Additionally, for TFIID/SAGA subunits, we identified 118/102 unique phosphorylated and 54/61 unique lysine acetylated sites. Especially, several lysine residues on the SAGA subunits Spt7p and Sgf73p were found to be acetylated. Using a spectral counting approach, we found that the shared subunit TAF5p is phosphorylated to a significant greater extent in SAGA than in TFIID. Finally, we were able to map for the first time the cleavage site in Spt7p that is related to formation of the SAGA-like complex SLIK/SALSA. In general, our combination of tandem affinity enrichment, digestion with different proteases, extensive prefractionation and high-resolution LC-MS identifies a large number of PTMs of TFIID and SAGA/SLIK that might aid in future functional studies on these transcription factors.
基础转录因子 TFIID 和染色质修饰复合物 SAGA 具有几个共同的亚基，对于转录调控至关重要。在这里，我们描述了来自酵母的 TFIID 和 SAGA 上的翻译后修饰 (PTM) 的深入分析。我们采用了多种方法，使用高分辨率质谱 (LC-MS) 结合胰蛋白酶、糜蛋白酶和 Glu-C 蛋白酶进行分析。累积的肽鉴定数据，假发现率<1%，使我们能够近乎完整地覆盖大多数 TFIID 和 SAGA 亚基序列。此外，对于 TFIID/SAGA 亚基，我们鉴定出了 118/102 个独特的磷酸化和 54/61 个独特的赖氨酸乙酰化位点。特别是，SAGA 亚基 Spt7p 和 Sgf73p 上的几个赖氨酸残基被发现乙酰化。使用光谱计数方法，我们发现共享亚基 TAF5p 在 SAGA 中的磷酸化程度明显高于 TFIID。最后，我们首次能够映射到 Spt7p 中的切割位点，该位点与 SAGA 样复合物 SLIK/SALSA 的形成有关。总的来说，我们的串联亲和富集、不同蛋白酶的消化、广泛的预分级和高分辨率 LC-MS 的组合，鉴定出了大量 TFIID 和 SAGA/SLIK 的 PTM，这可能有助于未来对这些转录因子的功能研究。
