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Amino Acid Mass Spectrometry Based on Polarity-Reversing Nano-Electrospray Ionization
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摘要: 氨基酸分析一直是化学和生物学分析研究的重要内容，在众多氨基酸分析方法中，质谱法在灵敏度和准确度上具有较大优势。液质联用是目前最为常用的氨基酸质谱分析方法，需要使用到电喷雾离子源。然而，电喷雾离子源对氨基酸离子化效率有限，尤其是酸性氨基酸，这严重影响了氨基酸质谱分析方法检测灵敏度的进一步提升。基于自主搭建的极性反转纳喷雾离子源（PR-nESI），建立了20种常见氨基酸的质谱检测方法。与基于普通纳喷雾离子源（nano-ESI）的检测方法相比，PR-nESI显著提高了检测信噪比，提升幅度最高可达数十倍。此外，PR-nESI具有较强的除盐效果，能有效抑制氨基酸金属离子加合物的形成，极大地方便了后期谱图指认。该氨基酸检测方法未来在临床、食品等领域中具有较为广阔的应用前景。Abstract: Amino acid analysis has always been a crucial aspect of chemical and biological research due to its essential biological significance and diverse roles in living organisms, including catalyzing chemical reactions, signaling, and supporting cell structure. Mass spectrometry possesses significant advantages in terms of sensitivity and accuracy among various amino acid analysis methods. Liquid chromatography-mass spectrometry (LC-MS) is the most commonly used method for amino acid mass spectrometry analysis, requiring an electrospray ion source. While the electrospray ion source has many advantages, such as high sensitivity, high selectivity, simple operation, compatibility with various types of mass spectrometers, and a wide range of analyzable substances, its ionization efficiency for amino acids is limited, particularly for acidic amino acids. This limitation hinders further improvement of the detection sensitivity of analytical methods. A mass spectrometry method for determining 20 common amino acids was established based on a self-developed Polarity-Reversing Nano-Electrospray Ionization (PR-nESI) ion source. PR-nESI enhances detection by altering the high-voltage strategy during the nano-spray process. Compared to conventional Nano-Electrospray Ionization (nano-ESI) methods, PR-nESI significantly improves the detection signal-to-noise ratio, increasing up to tens of times. Moreover, PR-nESI exhibits strong desalting effects, effectively inhibiting the formation of metal ion adducts of amino acids, which significantly facilitates spectral identification, particularly for biomolecules in salt buffer systems. The amino acid detection method presented in this study holds promising future application prospects in clinical and food fields.
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Key words:
- metrology /
- amino acid /
- mass spectrometry /
- polarity-reversing nano-electrospray ionization /
- nano-spray /
- desalting
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图 1 不同负高压时间的提取离子流图
Figure 1. The extracted ion chromatograms of isoleucine with varying durations of negative high pressure (1, 3, 6, and 9 s)
图 2 Ile的PR-nESI质谱图
Figure 2. Mass spectra of 30 μmol/L pure aqueous solutions of isoleucine (Ile) in positive ion mode under PR-nESI
图 4 不同负电压下PR-nESI 对异亮氨酸检测所得的S/N
Figure 4. Signal-to-noise ratio (S/N) of isoleucine detected by PR-nESI under different negative voltage settings
图 5 正离子模式下30 μmol/L Asp、Pro和Lys 纯水溶液在nano-ESI和PR-nESI下的质谱图
Figure 5. Mass spectra of 30 μmol/L pure aqueous solutions of aspartic acid (Asp), proline (Pro), and lysine (Lys) in positive ion mode under nano-ESI and PR-nESI
图 6 加入1 mmol/L NaCl后Asp、Pro和Lys 溶液在nano-ESI和PR-nESI下的质谱图
Figure 6. Mass spectra of aspartic acid (Asp), proline (Pro), and lysine (Lys) solutions under nano-ESI and PR-nESI after the addition of 1 mmol/L sodium chloride (NaCl)
表 1 nano-ESI和PR-nESI对20种氨基酸的平均检测S/N及比值
Table 1. Average detection signal-to-noise ratio (S/N) and ratios for 20 amino acids analyzed using nano-ESI and PR-nESI
氨基酸分类 氨基酸名称 S/N 比值 nano-ESI PR-nESI PR-nESI/nano-ESI 酸性氨基酸 天冬氨酸 1.16 203.53 174.7 谷氨酸 2.71 333.54 123.0 中性氨基酸 半胱氨酸 8.43 154.70 18.3 天冬酰胺 10.84 125.19 11.6 苯丙氨酸 40.92 132.42 3.2 谷氨酰胺 13.68 211.87 15.5 酪氨酸 29.16 159.13 5.5 丝氨酸 16.54 119.03 7.2 甲硫氨酸 39.22 157.69 4.0 色氨酸 43.57 171.49 3.9 缬氨酸 54.01 191.58 3.6 甘氨酸 14.30 145.28 10.2 亮氨酸 8.10 92.28 11.4 丙氨酸 11.95 99.83 8.4 异亮氨酸 30.32 162.00 5.3 苏氨酸 13.73 173.20 12.6 脯氨酸 24.57 124.22 5.1 碱性氨基酸 组氨酸 463.14 592.00 1.3 赖氨酸 484.30 578.66 1.2 精氨酸 435.80 504.37 1.2 
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[1] Ohno H, Fukumoto K. Amino acid ionic liquids.[J]. Accounts of Chemical Research, 2007, 40(11): 1122. doi: 10.1021/ar700053z [2] Liu J, Poojary M M, Zhu L, et al. Phenolic Acid–Amino Acid Adducts Exert Distinct Immunomodulatory Effects in Macrophages Compared to Parent Phenolic Acids[J]. Food Chem, 2023, 71(5): 2344-2355. doi: 10.1021/acs.jafc.2c06658 [3] 吴雪, 薄梦, 孙晓璟, 等. 液相色谱-质谱法检测甲磺酸沙非胺中基因毒性杂质[J]. 计量科学与技术, 2022, 66(11): 27-30. doi: 10.12338/j.issn.2096-9015.2021.0632 [4] 汤逸飞, 屠雨晨, 黄芳, 等. 基于QuEChERS-气相色谱三重四级杆串联质谱内标法测定结球甘蓝中30种农药残留的不确定度评定[J]. 计量科学与技术, 2022, 66(2): 25-32,8. [5] 张再平, 王玉. 电感耦合等离子体质谱法测定大米粉中砷、镉含量及不确定度评估[J]. 计量科学与技术, 2021, 65(12): 60-65. doi: 10.12338/j.issn.2096-9015.2019.0427 [6] 王许欣, 李晓敏, 张庆合. 不同定量方法在婴儿配方奶粉中维生素B12测量中的不确定度评估[J]. 计量科学与技术, 2022, 66(9): 23-27,46. doi: 10.12338/j.issn.2096-9015.2022.0089 [7] Kamleh, Anas M, Snowden, et al. LC-MS Metabolomics of Psoriasis Patients Reveals Disease Severity-Dependent Increases in Circulating Amino Acids That Are Ameliorated by Anti-TNF alpha Treatment[J]. Proteome Res, 2015, 14(1): 557-566. doi: 10.1021/pr500782g [8] 余利星, 翟睿, 龚晓云, 等. 基于磁性纳米材料分离的肿瘤标志物HSP90α质谱分析方法[J]. 计量技术, 2020(5): 14-18,78. [9] 金涛, 潘本松, 聂洪港. 基质辅助激光解吸电离-质谱成像技术(MALDI-MSI)及其在脑神经性疾病研究中的应用[J]. 计量科学与技术, 2022, 66(3): 3-22. [10] Vinayavekhin N, Homan E A, Saghatelian A. Exploring Disease through Metabolomics[J]. Acs Chemical Biology, 2010, 5(1): 91-103. doi: 10.1021/cb900271r [11] Liao H W, Chen G Y, Wu M S, et al. Development of a Postcolumn Infused-Internal Standard Liquid Chromatography Mass Spectrometry Method for Quantitative Metabolomics Studies[J]. Journal of Proteome Research, 2017, 16(2): 1097-1104. doi: 10.1021/acs.jproteome.6b01011 [12] 赵春霞, 许国旺. 基于液相色谱-质谱技术的代谢组学分析方法新进展[J]. 分析科学学报, 2014, 30(5): 761-766. doi: 10.13526/j.issn.1006-6144.2014.05.030 [13] Hollenberg J L, Ifft J B. Hydration numbers by near-infrared spectrophotometry. 1. Amino acids[J]. Journal of Physical Chemistry, 1982, 86(11): 1938-1941. doi: 10.1021/j100208a007 [14] Gui M, Rutan S C, Agbodjan A. Kinetic Detection of Overlapped Amino Acids in Thin-Layer Chromatography with a Direct Trilinear Decomposition Method[J]. Analytical Chemistry, 1995, 67(18): 3293-3299. doi: 10.1021/ac00114a028 [15] Brunauer L S, Caslavka K E, Groningen K V. Ion Exchange and Thin Layer Chromatographic Separation and Identification of Amino Acids in a Mixture: An Experiment for General Chemistry and Biotechnology Laboratories[J]. Journal of Chemical Education, 2014, 91(12): 2216-2220. doi: 10.1021/ed500226q [16] Syu K Y, Lin C L, Huang H C, et al. Determination of theanine, GABA, and other amino acids in green, oolong, black, and Pu-erh teas with dabsylation and high-performance liquid chromatography[J]. Journal of Agricultural & Food Chemistry, 2008, 56(17): 7637-7643. [17] 林丽敏, 张建莹, 肖锋, 等. 非衍生化/液相色谱-串联质谱法测定果蔬中氨基酸类有机磷除草剂残留[J]. 分析测试学报, 2022, 41(11): 1702-1706. doi: 10.19969/j.fxcsxb.22042805 [18] Mitsuru T, Keishiro A, Tomotaka T, et al. Laser Desorption Ionization-Mass Spectrometry with Graphite Carbon Black Nanoparticles for Simultaneous Detection of Taste- and Odor-Active Compounds[J]. ACS Appl. Nano Mater, 2022, 5(2): 2187-2194. doi: 10.1021/acsanm.1c03890 [19] Masataka Wakayama, Naohiro Aoki, Haruto Sasaki, et al. Simultaneous Analysis of Amino Acids and Carboxylic Acids by Capillary Electrophoresis−Mass Spectrometry Using an Acidic Electrolyte and Uncoated Fused-Silica Capillary[J]. Analytical Chemistry, 2010, 82(24): 9967-9976. doi: 10.1021/ac1019039 [20] 谭思源, 李曼莉, 傅博强, 等. 单细胞质谱分析方法研究进展[J]. 计量科学与技术, 2021, 65(5): 20-29,13. doi: 10.12338/j.issn.2096-9015.2020.9021 [21] 李佳乐, 武利庆, 金有训, 等. 人血清白蛋白纯品含量的同位素稀释质谱方法研究[J]. 计量学报, 2016, 37(3): 328-332. doi: 10.3969/j.issn.1000-1158.2016.03.022 [22] 苏福海. 《液相色谱-飞行时间质谱联用仪性能测定方法》国家标准解读[J]. 计量科学与技术, 2020(10): 3-5. doi: 10.3969/j.issn.2096-9015.2020.10.01 [23] 朱文, 倪鑫茹, 徐昇. 同位素稀释质谱法在蛋白质计量比对中的应用[J]. 计量与测试技术, 2022, 49(7): 12-14. doi: 10.15988/j.cnki.1004-6941.2022.7.005 [24] Pandey R , Collins M , Lu X , et al. Novel Strategy for Untargeted Chiral Metabolomics using Liquid Chromatography-High Resolution Tandem Mass Spectrometry[J]. Analytical chemistry, 93(14): 5805-5814. [25] 易可可, 谢洁, 龚晓云, 等. 液相色谱-串联质谱应用研究进展[J]. 计量科学与技术, 2021(2): 7-15,6. [26] Feng L L, Gong X Y, Song J F, et al. Strong Acid Anions Significantly Increasing the Charge State of Proteins during Electrospray Ionization[J]. Anal. Chem, 2020, 92(2): 1770-1779. doi: 10.1021/acs.analchem.9b03416 [27] Emmett M R, Caprioli R M. Micro-electrospray mass spectrometry: ultra-high-sensitivity analysis of peptides and proteins[J]. Journal of the American Society for Mass Spectrometry, 1994, 5(7): 605-613. doi: 10.1016/1044-0305(94)85001-1 [28] Wilm M S, Mann M. Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last? International Journal of Mass Spectrometry & Ion Processes, 1994, 136(2-3): 167-180. [29] Wei Z W, Xiong X C, Guo C G, et al. Pulsed Direct Current Electrospray: Enabling Systematic Analysis of Small Volume Sample by Boosting Sample Economy[J]. Analytical Chemistry, 2015, 87(22): 11242-11248. doi: 10.1021/acs.analchem.5b02115 [30] Nishant C, Catherine A C, David B G, et al. High-frequency AC electrospray ionization source for mass spectrometry of biomolecules[J]. Journal of the American Society for Mass Spectrometry, 2010, 21(11): 1852-1856. doi: 10.1016/j.jasms.2010.06.023 [31] Gong X Y, Xiong X C, Zhao Y C, et al. Boosting the Signal Intensity of Nanoelectrospray Ionization by Using a Polarity-Reversing High-Voltage Strategy[J]. Anal. Chem, 2017, 89(13): 7009-7016. doi: 10.1021/acs.analchem.7b00555 -
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