Mass Spectrometry Engineering Technology Research Center, Center for Advanced Measurement Science , National Institute of Metrology , Beijing 100029 , People's Republic of China.
Anal Chem. 2019 Feb 5;91(3):1826-1837. doi: 10.1021/acs.analchem.8b02759. Epub 2019 Jan 23.
Supercharging is beneficial in many ways to the analysis of proteins by mass spectrometry (MS). In this work, a novel supercharging method was developed. It made use of our previously developed ionization technique: namely, polarity reversed nanoelectrospray ionization (PR-nESI) for the ionization of proteins. Supercharging of proteins was achieved by just adding 1-10 mM of a salt to the sample, such as sodium chloride (NaCl). The charge state of proteins obtained by our method was significantly higher than that by nano-ESI with 1% (v/v) acetic acid (HAc). Different kinds of salts were investigated. Salts with strong acid anions were capable of supercharging proteins, including chlorides, bromides, iodides, and nitrates. The signal intensity and signal to noise ratio ( S/ N) of proteins were increased at the same time. Phosphates were also found to have a supercharging effect, due to the fact that phosphoric acid was a medium-strong acid. In comparison, salts with weak acid anions had no supercharging effect, such as carbonates, sulfides, acetates, and formates. The species of the salt anion was critical to the supercharging effect, while the species of the salt cation showed little influence on the supercharging effect. Investigations were made into the mechanism of our method. The supercharging effect was caused by interactions between protein molecules and salt anions, as well as the influence of protons. The present work offered us an alternative way for the supercharging of proteins. The use of common salts for supercharging made the procedure more convenient. The concentration of salts needed for supercharging was much lower than those conventionally used for supercharging reagents. Taking into consideration the fact that many biological samples are buffered with phosphates and chlorides, these samples could be directly supercharged by our method without any additional additives. Furthermore, as many salts are nontoxic and can easily be found in a chemical laboratory, the use of salts for supercharging would be a much more practical and economical choice. In addition, the present work also furthered our understandings about the mechanism of supercharging, as well as electrospray.
电喷雾离子化（ESI）是目前应用最广泛的质谱（MS）样品离子化技术之一。然而，ESI 过程中形成的多电荷态离子的效率通常较低，这限制了 MS 对低丰度蛋白质的检测能力。为了克服这一限制，人们开发了多种增强离子化效率的技术，例如电喷雾辅助解吸电离（DESI）和基质辅助激光解吸电离（MALDI）。这些技术虽然可以提高蛋白质的检测灵敏度，但它们的应用范围和效率仍然存在一定的局限性。
在本研究中，我们开发了一种新的增强蛋白质电喷雾离子化效率的方法，即盐增强电喷雾离子化（SE-ESI）。该方法利用了我们之前开发的极性反转纳米电喷雾离子化（PR-nESI）技术，通过在样品中添加 1-10 mM 的盐（如氯化钠（NaCl））来实现蛋白质的多电荷态离子化。与传统的 ESI 相比，我们的方法可以显著提高蛋白质的电荷状态，同时提高蛋白质的信号强度和信噪比（S/N）。
我们研究了不同类型的盐对蛋白质 SE-ESI 的影响。结果表明，具有强酸阴离子的盐（如氯化物、溴化物、碘化物和硝酸盐）可以增强蛋白质的离子化效率，而具有弱酸阴离子的盐（如碳酸盐、硫化物、醋酸盐和甲酸盐）则没有增强效果。此外，我们还发现磷酸盐也具有增强效果，这是因为磷酸是一种中强酸。
盐阴离子的种类对 SE-ESI 效果至关重要，而盐阳离子的种类则影响较小。我们进一步研究了 SE-ESI 的机制，发现蛋白质分子与盐阴离子之间的相互作用以及质子的影响是导致 SE-ESI 增强效果的主要原因。
本研究为蛋白质的多电荷态离子化提供了一种新的方法，利用常见的盐作为增强剂可以使实验操作更加简便。与传统的增强剂相比，我们的方法所需盐的浓度更低。此外，由于许多生物样品中都含有磷酸盐和氯化物等缓冲盐，因此可以直接使用我们的方法对这些样品进行增强，无需添加额外的添加剂。
总之，我们的研究结果表明，盐增强电喷雾离子化是一种简单、高效、实用的蛋白质离子化方法，具有广泛的应用前景。
