Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA.
Resource for Native Mass Spectrometry Guided Structural Biology, The Ohio State University, Columbus, OH, USA.
Nat Protoc. 2020 Mar;15(3):1132-1157. doi: 10.1038/s41596-019-0281-0. Epub 2020 Jan 31.
It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.
在蛋白质纯化过程中和之后，评估蛋白质和蛋白质复合物的身份和纯度非常重要，以确保样品具有足够的质量，可用于进一步的生化和结构特征分析，以及用于消费品、化学过程和治疗学。由于其在测量过程中保留非共价相互作用的能力，因此，天然质谱（nMS）已成为蛋白质分析的重要工具，使其能够以高灵敏度和高速获得蛋白质结构信息。非挥发性物质的存在会产生干扰，通常通过离线缓冲交换来缓解，但这种方法既耗时又难以自动化。我们提供了一种快速在线缓冲交换（OBE）nMS 的方案，可直接筛选预纯化蛋白质、蛋白质复合物或澄清细胞裂解物的结构特征。在本方案中描述的液相色谱-质谱（LC-MS）方法中，将处于 MS 不兼容条件下的样品注入短尺寸排阻色谱柱。使用水性、非变性流动相从小分子非挥发性缓冲成分中分离蛋白质和蛋白质复合物。在电喷雾电离后，通过质谱仪检测洗脱的蛋白质和蛋白质复合物。质谱可以提供有关蛋白质样品纯度和寡聚化的信息，并且额外的串联质谱可以帮助进一步获得有关蛋白质复合物亚基的信息。通过 OBE nMS 获得的信息可用于快速（<5 分钟）质量控制，并可进一步指导蛋白质表达和纯化优化。
