Hadavi Darya, Ng Che Yee, Zhao Yuandi, Mathew Anjusha, Anthony Ian G M, Cillero-Pastor Berta, Cuypers Eva, Siegel Tiffany Porta, Honing Maarten
Maastricht Multi Modal Molecular Imaging (M4i) Institute, Division of Imaging Mass Spectrometry (IMS), Maastricht University, Maastricht, The Netherlands.
MERLN Institute for Technology-Inspired Regenerative Medicine, Department of Cell Biology-Inspired Tissue Engineering (cBITE), Maastricht University, Maastricht, The Netherlands.
Rapid Commun Mass Spectrom. 2025 Jul 30;39(14):e10048. doi: 10.1002/rcm.10048.
To perform native mass spectrometry (MS) studies, there are a limited number of volatile and electrospray ionization (ESI)-MS compatible solutions, such as ammonium bicarbonate and ammonium acetate (AA). These solutions could induce the unfolding of proteins due to the formation of CO bubbles or induced acidification during ESI. Hence, it was important to introduce a buffer suitable to preserve the native form of proteins while simulating physiological conditions.
The 4-ethylmorpholinium/acetate (4EM/A) buffer was compared to AA for the analysis of proteins and protein complexes with mass ranges from 5 to 103 kDa and isoelectric points (pI) between 3 and 11. The evaluations were conducted by comparing the native-MS profiles, CCS values, arrival time distributions (ATDs), and proteins bioactivities. The human cardiac troponin complex (cTn complex) and its subunit cardiac troponin T (cTnT) were analyzed as proof of the applicability of this buffer for challenging proteins and protein complexes.
4EM/A led to lower charge states compared to AA, supporting the likelihood of preserving protein folding during nano-ESI and in a high vacuum environment of MS. Ion mobility measurements revealed that proteins in 4EM/A exhibit a lower degree of conformational variation compared to AA, suggesting enhanced conformational stability and potential retention of natural-like compactness. Additionally, testing the impact of 4EM/A on bioactivity, lysozyme showed increased biological activity in 4EM/A relative to AA, highlighting the buffer's potential for real-time assessment of protein interaction kinetics and bioactivity. The 4EM/A buffer enabled native-MS analysis of cTnT for the first time.
We introduced 4EM/A, with pK of 7.72/4.76, as a promising buffer for native-MS studies to maintain protein and protein complex bioactivity and conformational integrity.
为了进行天然质谱（MS）研究，挥发性且与电喷雾电离（ESI）-MS兼容的溶液数量有限，例如碳酸氢铵和醋酸铵（AA）。这些溶液可能会因在ESI过程中形成CO气泡或导致酸化而促使蛋白质展开。因此，引入一种适合在模拟生理条件下保存蛋白质天然形式的缓冲液非常重要。
将4-乙基吗啉/醋酸盐（4EM/A）缓冲液与AA进行比较，用于分析质量范围为5至103 kDa且等电点（pI）在3至11之间的蛋白质和蛋白质复合物。通过比较天然MS图谱、CCS值、到达时间分布（ATD）和蛋白质生物活性来进行评估。分析人心脏肌钙蛋白复合物（cTn复合物）及其亚基心脏肌钙蛋白T（cTnT），以证明该缓冲液对具有挑战性的蛋白质和蛋白质复合物的适用性。
与AA相比，4EM/A导致的电荷态更低，这支持了在纳米ESI和MS的高真空环境中保留蛋白质折叠的可能性。离子淌度测量表明，与AA相比，4EM/A中的蛋白质构象变化程度更低，表明构象稳定性增强且可能保留了类似天然的紧密性。此外，测试4EM/A对生物活性的影响时，溶菌酶在4EM/A中相对于AA显示出更高的生物活性，突出了该缓冲液在实时评估蛋白质相互作用动力学和生物活性方面的潜力。4EM/A缓冲液首次实现了对cTnT的天然MS分析。
我们引入了pK为7.72/4.76的4EM/A，作为用于天然MS研究的有前景的缓冲液，以维持蛋白质和蛋白质复合物的生物活性及构象完整性。
