National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
Rapid Commun Mass Spectrom. 2022 Nov 15;36(21):e9395. doi: 10.1002/rcm.9395.
Native mass spectrometry (MS) is an analytical technique used to determine the molecular mass of protein complexes without cross-linking. Size exclusion chromatography (SEC) coupled with native MS using conventional electrospray ionization (ESI) has been reported to allow online buffer exchange. To detect a wide variety of protein complexes without a collapse in the ionization process, it is important to build an online system that enables robust analysis with a low flow rate.
We created an online native MS system equipped with nanoESI connected to the SEC component (online SEC/nanoESI system) and optimized several parameters for SEC separation and ionization. The constructed system was used to measure a solution consisting of a protein mixture of various molecular masses (10-300 kDa) to verify characteristics such as the measurable molecular mass range, reproducibility, and online buffer exchange.
The optimal flow rates for SEC separation and nanoESI analysis using this system were 200 and 1 μL/min, respectively. This system was able to analyze proteins in the ranges of 10-300 and 20-300 kDa for protein samples in ammonium acetate and nonvolatile buffer, respectively. Furthermore, the results of consecutive measurements showed that the relative standard deviations of the retention times and observed masses for each protein were sufficiently small.
We created an online SEC/nanoESI system and evaluated its utility for the analysis of various proteins in conventional measurement solvent and nonvolatile buffer. As a result, the structural stability and resolution of the proteins were found to be sufficient when using online buffer exchange. Therefore, this online SEC/nanoESI system would be a useful technique for obtaining mass spectra of various proteins automatically with good resolution, simply by loading samples into an autosampler.
天然质谱（MS）是一种分析技术，用于在不交联的情况下确定蛋白质复合物的分子量。已报道将尺寸排阻色谱（SEC）与使用常规电喷雾电离（ESI）的天然 MS 耦合，以允许在线缓冲交换。为了在不使电离过程崩溃的情况下检测各种蛋白质复合物，重要的是构建一个能够以低流速进行稳健分析的在线系统。
我们创建了一个配备有与 SEC 组件相连的纳喷雾（nanoESI）的在线天然 MS 系统（在线 SEC/nanoESI 系统），并优化了 SEC 分离和电离的几个参数。该构建的系统用于测量由各种分子量的蛋白质混合物组成的溶液（10-300 kDa），以验证可测量的分子量范围、重现性和在线缓冲交换等特性。
该系统用于 SEC 分离和 nanoESI 分析的最佳流速分别为 200 和 1 μL/min。该系统能够分析在乙酸铵和非挥发性缓冲液中蛋白质样品的分子量范围分别为 10-300 和 20-300 kDa 的蛋白质。此外，连续测量的结果表明，每个蛋白质的保留时间和观察到的质量的相对标准偏差足够小。
我们创建了一个在线 SEC/nanoESI 系统，并评估了其在常规测量溶剂和非挥发性缓冲液中分析各种蛋白质的实用性。结果表明，当使用在线缓冲交换时，蛋白质的结构稳定性和分辨率足够好。因此，该在线 SEC/nanoESI 系统将是一种有用的技术，可通过简单地将样品加载到自动进样器中，自动获得各种蛋白质的良好分辨率的质谱图。
