Reddy B Sudarshana, Chary V Naresh, Pavankumar P, Prabhakar S
Academy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500 007, Telangana, India.
National Centre for Mass Spectrometry, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500 007, Telangana, India.
J Mass Spectrom. 2016 Aug;51(8):638-650. doi: 10.1002/jms.3788.
Methylation is an essential metabolic process in the biological systems, and it is significant for several biological reactions in living organisms. Methylated compounds are known to be involved in most of the bodily functions, and some of them serve as biomarkers. Theoretically, all α-amino acids can be methylated, and it is possible to encounter them in most animal/plant samples. But the analytical data, especially the mass spectral data, are available only for a few of the methylated amino acids. Thus, it is essential to generate mass spectral data and to develop mass spectrometry methods for the identification of all possible methylated amino acids for future metabolomic studies. In this study, all N-methyl and N,N-dimethyl amino acids were synthesized by the methylation of α-amino acids and characterized by a GC-MS method. The methylated amino acids were derivatized with ethyl chloroformate and analyzed by GC-MS under EI and methane/CI conditions. The EI mass spectra of ethyl chloroformate derivatives of N-methyl (1-18) and N,N-dimethyl amino acids (19-35) showed abundant [M-COOC H ] ions. The fragment ions due to loss of C H , CO , (CO + C H ) from [M-COOC H ] were of structure indicative for 1-18. The EI spectra of 19-35 showed less number of fragment ions when compared with those of 1-18. The side chain group (R) caused specific fragment ions characteristic to its structure. The methane/CI spectra of the studied compounds showed [M + H] ions to substantiate their molecular weights. The detected EI fragment ions were characteristic of the structure that made easy identification of the studied compounds, including isomeric/isobaric compounds. Fragmentation patterns of the studied compounds (1-35) were confirmed by high-resolution mass spectra data and further substantiated by the data obtained from C -labeled glycines and N-ethoxycarbonyl methoxy esters. The method was applied to human plasma samples for the identification of amino acids and methylated amino acids. Copyright © 2016 John Wiley & Sons, Ltd.
甲基化是生物系统中一种重要的代谢过程，对生物体中的多种生物反应具有重要意义。已知甲基化化合物参与了大多数身体机能，其中一些还可作为生物标志物。理论上，所有α-氨基酸都可以被甲基化，并且在大多数动植物样本中都可能出现。但是，目前仅获得了少数甲基化氨基酸的分析数据，尤其是质谱数据。因此，为了未来的代谢组学研究，生成质谱数据并开发用于鉴定所有可能的甲基化氨基酸的质谱方法至关重要。在本研究中，通过α-氨基酸的甲基化反应合成了所有N-甲基和N,N-二甲基氨基酸，并采用气相色谱-质谱联用（GC-MS）方法对其进行了表征。甲基化氨基酸用氯甲酸乙酯进行衍生化处理，并在电子轰击电离（EI）和甲烷/化学电离（CI）条件下通过GC-MS进行分析。N-甲基氨基酸（1-18）和N,N-二甲基氨基酸（19-35）的氯甲酸乙酯衍生物的EI质谱显示出丰富的[M-COOC₂H₅]离子。从[M-COOC₂H₅]离子中失去C₂H₄、CO、（CO₂ + C₂H₄）产生的碎片离子具有指示1-18结构的特征。与1-18相比，19-35的EI谱图显示出较少的碎片离子。侧链基团（R）产生了具有其结构特征的特定碎片离子。所研究化合物的甲烷/CI谱图显示出[M + H]离子，证实了它们的分子量。检测到的EI碎片离子具有易于鉴定所研究化合物（包括异构体/等压异构体）结构的特征。所研究化合物（1-35）的裂解模式通过高分辨率质谱数据得到证实，并进一步由¹³C标记的甘氨酸和N-乙氧基羰基甲氧基酯的数据得到验证。该方法应用于人体血浆样本中氨基酸和甲基化氨基酸的鉴定。版权所有© 2016约翰威立父子有限公司。
