Olinares Paul Dominic B, Chait Brian T
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY, USA.
Methods Mol Biol. 2020;2062:357-382. doi: 10.1007/978-1-4939-9822-7_17.
Native mass spectrometry (MS) enables direct mass measurement of intact protein assemblies generating relevant subunit composition and stoichiometry information. Combined with cross-linking and structural data, native MS-derived information is crucial for elucidating the architecture of macromolecular assemblies by integrative structural methods. The exosome complex from budding yeast was among the first endogenous protein complexes to be affinity isolated and subsequently characterized by this technique, providing improved understanding of its composition and structure. We present a protocol that couples efficient affinity capture of yeast exosome complexes and sensitive native MS analysis, including rapid affinity isolation of the endogenous exosome complex from cryolysed yeast cells, elution in nondenaturing conditions by protease cleavage, depletion of the protease, buffer exchange, and native MS measurements using an Orbitrap-based instrument (Exactive Plus EMR).
原生质谱（MS）能够直接对完整的蛋白质组装体进行质量测量，生成相关的亚基组成和化学计量信息。结合交联和结构数据，原生质谱衍生的信息对于通过综合结构方法阐明大分子组装体的结构至关重要。来自芽殖酵母的外泌体复合物是最早通过亲和分离并随后用该技术表征的内源性蛋白质复合物之一，这有助于更好地理解其组成和结构。我们提出了一种方案，该方案将酵母外泌体复合物的高效亲和捕获与灵敏的原生质谱分析相结合，包括从冷冻裂解的酵母细胞中快速亲和分离内源性外泌体复合物、通过蛋白酶切割在非变性条件下洗脱、去除蛋白酶、缓冲液交换以及使用基于轨道阱的仪器（Exactive Plus EMR）进行原生质谱测量。
