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首页> 外文期刊>Journal of mass spectrometry: JMS >CTP synthetase activity assay by liquid chromatography tandem mass spectrometry in the multiple reaction monitoring mode
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CTP synthetase activity assay by liquid chromatography tandem mass spectrometry in the multiple reaction monitoring mode

机译：CTP合成酶活性测定通过液相色谱串联质谱法测定多反应监测模式

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Cytidine 5 '-triphosphate synthetase (CTPS) is known to be a central enzyme in the de novo synthesis of CTP. We have recently demonstrated that a deficiency in CTPS1 is associated with an impaired capacity of activated lymphocytes to proliferate leading to a combined immunodeficiency disease. In order to better document its role in immunomodulation, we developed a method for measuring CTPS activity in human lymphocytes. Using liquid chromatography-mass spectrometry, we quantified CTPS activity by measuring CTP in cell lysates. A stable isotope analog of CTP served as internal standard. We characterized the kinetic parameters V-max and K-m of CTPS and verified that an inhibition of the enzyme activity was induced after 3-deazauridine (3DAU) treatment, a known inhibitor of CTPS. We then determined CTPS activity in healthy volunteers, in a family whose child displayed a homozygous mutation in CTPS1 gene and in patients who had developed or not a chronic lung allograft dysfunction (CLAD) after lung transplantation. Linearity of the CTP determination was observed up to 451 mu mol/L, with accuracy in the 15% tolerance range. Michaelis-Menten kinetics for lysates of resting cells were K-m=280 +/- 310 mu mol/L for UTP, V-max=83 +/- 20 pmol/min and, for lysates of activated PBMCs, K-m=230 +/- 280 mu mol/L for UTP, V-max=379 +/- 90 pmol/min. Treatment by 3DAU and homozygous mutation in CTPS1 gene abolished the induction of CTPS activity associated with cell stimulation, and CTPS activity was significantly reduced in the patients who developed CLAD. We conclude that this test is suitable to reveal the involvement of CTPS alteration in immunodeficiency.
机译：已知胞嘧啶5'-三磷酸合成酶（CTPS）是DE Novo合成CTP中的中枢酶。我们最近证明CTPS1的缺陷与活性淋巴细胞的容量受损，以扩散导致综合免疫缺陷疾病。为了更好地记录其在免疫调节中的作用，我们开发了一种测量人淋巴细胞中CTP活性的方法。使用液相色谱 - 质谱法，通过测量细胞裂解物中的CTP来定量CTPS活性。 CTP的稳定同位素类似物作为内标。我们的特征在于CTP的动力学参数V-MAX和K-M，并验证诱导酶活性的抑制在3-二氮胺（3DAU）处理后，已知的CTP抑制剂。然后，我们在健康志愿者中确定了CTPS活动，在儿童在CTPS1基因中显示出纯合突变的家庭中，并且在肺移植后开发或非慢性肺同种异体移植功能障碍（包层）的患者中。 CTP测定的线性测定高达451μmol/ L，在15％的公差范围内精确度。用于休息细胞的裂解物的Michaelis-Menten动力学均为HIM = 280 +/- 310 mm mol / l，V-max = 83 +/-20 pmol / min，对于活性PBMCs的裂解物，KM = 230 +/- UTP 280 mm mol / l，V-max = 379 +/- 90 pmol / min。 CTPS1基因中的3DAU和纯合突变治疗废除了与细胞刺激相关的CTPs活性的诱导，并且CTPS活性在开发包层的患者中显着降低。我们得出结论，该测试适用于揭示CTPS改变在免疫缺陷中的参与。

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