Stable isotope chemical labeling liquid chromatography-mass spectrometry (LC-MS) is a powerful strategy for comprehensive metabolomics profiling, which can improve metabolites coverage and quantitative information for exploration of metabolic regulation in complex biological systems. In the current work, a novel stable isotope N-phosphoryl amino acids labeling strategy (SIPAL) has been successful developed for quantitative profiling of amine-containing metabolites in urine based on organic phosphorus chemistry. Two isotopic reagents, 16O2- and 18O2-N-diisopropyl phosphoryl l-alanine N-hydroxysuccinimide esters (16O/18O-DIPP-L-Ala-NHS), were firstly synthesized in high yields for labeling the amine-containing metabolites. The performance of SIPAL strategy was tested by analyzing standard samples including 20 l-amino acids, 10 d-amino acids and small peptides by using LC-MS. We observed highly efficient and selective labeling for SIPAL strategy within 15 min in a one-pot derivatization reaction under aqueous reaction conditions. The introduction of a neutral phosphate group at N-terminus can increase the proton affinity and overall hydrophobicity of targeted metabolites, leading to the better ionization efficiency in electrospray ionization processes and chromatographic separations of hydrophilic metabolites on reversed-phase column. Furthermore, the chiral metabolites, such as d-amino acids, could be converted to diastereomers after SIPAL and successfully separated on regular reversed-phase column. The chirality of labeled enantiomers can be determined by using different detection methods such as 31P NMR, UV, and MS, demonstrating the potential application of SIPAL strategy. In addition, absolute quantification of chiral metabolites in biological samples can be easily achieved by using SIPAL strategy. For this purpose, urine samples collected from a healthy volunteer were analyzed by using LC-ESI-Orbitrap MS. Over 300 pairs of different amine-containing metabolites have been manually identified with high relative abundance (signal-to-noise ratios greater than 10). Finally, a standard peptide could be relatively quantified by using SIPAL strategy in combination with MALDI-TOF MS, suggesting the potential application of this strategy for quantitative proteomics.

中文翻译：

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使用液相色谱质谱法对含胺代谢物进行定量分析的稳定同位素 N-磷酰氨基酸标记

稳定同位素化学标记液相色谱-质谱 (LC-MS) 是一种强大的综合代谢组学分析策略，可以提高代谢物覆盖率和定量信息，以探索复杂生物系统中的代谢调控。在目前的工作中，一种新型稳定同位素 N-磷酰氨基酸标记策略 (SIPAL) 已成功开发，用于基于有机磷化学对尿液中含胺代谢物进行定量分析。两种同位素试剂 16O2- 和 18O2-N-二异丙基磷酰 l-丙氨酸 N-羟基琥珀酰亚胺酯 (16O/18O-DIPP-L-Ala-NHS) 首先以高产率合成，用于标记含胺的代谢物。通过分析包括 20 个 l-氨基酸的标准样品来测试 SIPAL 策略的性能，使用 LC-MS 分析 10 个 d-氨基酸和小肽。我们在水性反应条件下的一锅衍生反应中观察到 SIPAL 策略在 15 分钟内的高效和选择性标记。在 N 端引入中性磷酸基团可以增加目标代谢物的质子亲和力和整体疏水性，从而在反相柱上的电喷雾电离过程和亲水性代谢物的色谱分离中获得更好的电离效率。此外，手性代谢物，如 d-氨基酸，可以在 SIPAL 后转化为非对映异构体，并在常规反相柱上成功分离。标记对映异构体的手性可以通过使用不同的检测方法来确定，例如 31P NMR、UV 和 MS，展示 SIPAL 策略的潜在应用。此外，使用 SIPAL 策略可以轻松实现生物样品中手性代谢物的绝对定量。为此，使用 LC-ESI-Orbitrap MS 分析从健康志愿者收集的尿液样本。已经手动鉴定了超过 300 对不同的含胺代谢物，具有较高的相对丰度（信噪比大于 10）。最后，标准肽可以通过使用 SIPAL 策略与 MALDI-TOF MS 相结合进行相对定量，表明该策略在定量蛋白质组学中的潜在应用。使用 LC-ESI-Orbitrap MS 分析从健康志愿者收集的尿样。已经手动鉴定了超过 300 对不同的含胺代谢物，具有较高的相对丰度（信噪比大于 10）。最后，标准肽可以通过使用 SIPAL 策略与 MALDI-TOF MS 相结合进行相对定量，表明该策略在定量蛋白质组学中的潜在应用。使用 LC-ESI-Orbitrap MS 分析从健康志愿者收集的尿样。已经手动鉴定了超过 300 对不同的含胺代谢物，具有较高的相对丰度（信噪比大于 10）。最后，标准肽可以通过使用 SIPAL 策略与 MALDI-TOF MS 相结合进行相对定量，表明该策略在定量蛋白质组学中的潜在应用。