Ng Daniel H J, Chan Li Yan, Fitzner Laura, Keppler Julia Katharina, Ismail Shareef M, Hird Simon, Hancock Peter, Karin Schwarz, Tobias Demetrowitsch
International Food and Water Research Centre, Waters Pacific Pte Ltd, 1 Science Park Road #01-10, The Capricorn, Singapore Science Park II, Singapore, 117528, Singapore.
Division of Food Technology, Kiel University, Heinrich-Hecht Platz 10, Kiel, 24118, Germany.
Anal Methods. 2023 Jan 26;15(4):445-454. doi: 10.1039/d2ay01588a.
There are at least 500 naturally occurring amino acids, of which only 20 standard proteinogenic amino acids are used universally across all organisms in the synthesis of peptides and proteins. Non-standard amino acids can be incorporated into proteins or are intermediates and products of metabolic pathways. While the analysis of standard amino acids is well-defined, the analysis of non-standard amino acids can be challenging due to the wide range of physicochemical properties, and the lack of both reference standards and information in curated databases to aid compound identification. It has been shown that the use of an AccQ·Tag™ derivatization kit along with LC-MS/MS is an attractive option for the analysis of free standard amino acids in complex samples because it is fast, sensitive, reproducible, and selective. It has been demonstrated that the most abundant quantitative transition for MS/MS analysis of 6-aminoquinolyl--hydroxysuccinimidyl carbamate (AQC) derivatized amino acids corresponds to the fragmentation of the molecule at the 6-aminoquinoline carbonyl group producing a common / 171 fragment ion and occurs at similar mass spectrometry collision energy and cone voltages. In this study, the unique properties of AQC derivatized amino acids producing high intensity common fragment ions, along with chromatographic separation of amino acids under generic chromatography conditions, were used to develop a novel screening method for the detection of trace levels of non-standard amino acids in complex matrices. Structural elucidation was carried out by comparing the MS/MS fragment ion mass spectra generated with predicted fragmentation spectra to enable a putative identification, which was confirmed using an appropriate analytical standard. This workflow was applied to screen human plasma samples for bioactive thiol-group modified cysteine amino acids and -allylmercaptocysteine (SAMC), -allylcysteine sulfoxide (SACS or alliin) and -propenylcysteine (S1PC) are reported for the first time to be present in human plasma samples after the administration of garlic supplements.
天然存在的氨基酸至少有500种，其中只有20种标准的蛋白质ogenic氨基酸在所有生物体合成肽和蛋白质时被普遍使用。非标准氨基酸可被纳入蛋白质中，或作为代谢途径的中间体和产物。虽然标准氨基酸的分析方法已明确，但非标准氨基酸的分析可能具有挑战性，因为其物理化学性质范围广泛，且缺乏参考标准品以及经整理的数据库中有助于化合物鉴定的信息。研究表明，使用AccQ·Tag™衍生化试剂盒结合液相色谱-串联质谱（LC-MS/MS）是分析复杂样品中游离标准氨基酸的一个有吸引力的选择，因为它快速、灵敏、可重现且具有选择性。已证明，6-氨基喹啉-N-羟基琥珀酰亚胺基氨基甲酸酯（AQC）衍生化氨基酸的串联质谱分析中，最丰富的定量跃迁对应于分子在6-氨基喹啉羰基处的裂解，产生常见的m/z 171碎片离子，且在相似的质谱碰撞能量和锥电压下发生。在本研究中，利用AQC衍生化氨基酸产生高强度常见碎片离子的独特性质，以及在通用色谱条件下氨基酸的色谱分离，开发了一种用于检测复杂基质中痕量非标准氨基酸的新型筛查方法。通过将生成的串联质谱碎片离子质谱与预测的裂解质谱进行比较来进行结构解析，以实现推定鉴定，并使用适当的分析标准品进行确认。该工作流程应用于筛查人血浆样品中的生物活性巯基修饰的半胱氨酸氨基酸，首次报道在给予大蒜补充剂后人血浆样品中存在S-烯丙基半胱氨酸（SAMC）、S-烯丙基半胱氨酸亚砜（SACS或蒜氨酸）和S-丙烯基半胱氨酸（S1PC）。
