Small Emitter Tips for Native Mass Spectrometry of Proteins and Protein Complexes from Nonvolatile Buffers That Mimic the Intracellular Environment
Small Emitter Tips for Native Mass Spectrometry of Proteins and Protein Complexes from Nonvolatile Buffers That Mimic the Intracellular Environment
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DOI：
10.1021/acs.analchem.6b04897
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发表时间：
2017-03-07
影响因子：
7.4
通讯作者：
Williams, Evan R.
中科院分区：
文献类型：
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作者：
Susa, Anna C.;Xia, Zijie;Williams, Evan R.
盐通常是维持许多蛋白质和蛋白质复合物的天然结构和功能所必需的，但许多缓冲液对通过天然质谱（MS）进行的蛋白质分析产生不利影响。在此，蛋白质和蛋白质复合物离子直接由pH 7的150 mM KCl和25 mM Tris-HCl缓冲液形成，所述缓冲液广泛用于蛋白质化学以模拟细胞内环境。使用1.6微米直径的发射器尖端，蛋白质电荷状态分布没有从电喷雾电离MS中解析，导致没有质量信息。与此相反，电荷状态分布很好地解决了使用0.5 μ m的提示，从中可以获得的蛋白质和蛋白质复合物的质量。在-1.6 μ m以下，盐与蛋白质离子的加合随针尖尺寸的减小而减小，但在此尺寸以上则不减小。这表明，减少盐加合的机制是形成平均每个液滴少于一个蛋白质分子的小初始液滴，这降低了含有蛋白质分子的液滴中的盐：蛋白质比率。这是第一次证明蛋白质和蛋白质复合物离子的天然质谱分析，该蛋白质和蛋白质复合物离子由含有模拟细胞内环境的非挥发性盐的生理离子强度的缓冲液形成，并且该方法在质量分析之前或期间不需要样品制备或向蛋白质溶液中添加试剂。
Salts are often necessary to maintain the native structures and functions of many proteins and protein complexes, but many buffers adversely affect protein analysis by native mass spectrometry (MS). Here, protein and protein complex ions are formed directly from a 150 mM KCI and 2S mM Tris-HC1 buffer at pH 7 that is widely used in protein chemistry to mimic the intracellular environment. The protein charge-state distributions are not resolved from electrospray ionization MS using 1.6 ptm diameter emitter tips, resulting in no mass information. In contrast, the charge-state distributions are well-resolved using 0.5 mu m tips, from which the masses of proteins and protein complexes can be obtained. Adduction of salt to protein ions decreases with decreasing tip size below -1.6 mu m but not above this size. This suggests that the mechanism for reducing salt adduction is the formation of small initial droplets with on average fewer than one protein molecule per droplet, which lowers the salt:protein ratio in droplets that contain a protein molecule. This is the first demonstration of native mass spectrometry of protein and protein complex ions formed from a buffer containing physiological ionic strengths of nonvolatile salts that mimics the intracellular environment, and this method does not require sample preparation or addition of reagents to the protein solution before or during mass analysis.