Identification and quantification of low-molecular weight bacterial nucleotide signaling molecules by HPLC-coupled mass spectrometry including ion mobility technology

在过去的几年里，已经描述了许多低分子质量的细菌信号分子，要么存在于细胞质中，要么分泌出来。其中，线性核苷酸和环核苷酸在重要细菌功能的调节中起着第二信使的作用，即鸟苷3‘-二磷酸、5’-三磷酸(PppGpp)、鸟苷-3‘，5’-二焦磷酸(PpGpp)、3‘，5’-环腺苷一磷酸(CAMP)和3‘，5’-环鸟苷一磷酸(CGMP)、环二聚鸟苷3‘，5’-一磷酸(c-di-GMP)、环二聚腺苷3‘，5’-一磷酸(c-di-AMP)和环3‘，5’-鸟苷一磷酸(3，3‘-cAMP)。由于它们的生理浓度低，代谢速度快，鉴定和定量这些分子是一项极具挑战性的分析任务。我们开发了灵敏和特异的LC-MS/MS方法，主要专注于明确定量线性和环核苷酸、环二核苷酸及其各自的降解产物。在这种情况下，LC-MS/MS包括同位素标记的内标物、特定数量的分析和几个限定物的质量转变代表了所选择的分析方法。然而，必须特别注意样品的初始制备步骤、高效液相色谱方法的稳定性以及可靠的MS/MS程序。建立的程序可以很容易地适用于额外分子的分析，例如核苷酸类似物。本项目旨在进一步优化已建立的LC-MS/MS方法，包括进一步改进样品提取程序，建立细胞上清液中分泌信号分子的特异提取程序，以及利用重组酶生物合成同位素标记的内标。我们在LC-MS/MS技术方面的专业知识将作为一个Z单元与SPP 1879中的许多合作伙伴密切合作提供，特别是在细菌核苷酸信号分子的识别和量化方面。

Numerous low-molecular weight bacterial signaling molecules, either present in the cytosol or being secreted, have been described during the last years. Among them, linear as well as cyclic nucleotides play major roles as second messengers in the regulation of important bacterial functions, i.e. guanosine 3´-diphosphate, 5´-triphosphate (pppGpp), guanosine-3´,5´-bispyrophosphate (ppGpp), 3´,5´-cyclic adenosine monophosphate (cAMP) and 3´,5´-cyclic guanosine monophosphate (cGMP), cyclic dimeric guanosine 3´,5´-monophosphate (c-di-GMP), cyclic dimeric adenosine 3´,5´-monophosphate (c-di-AMP) and cyclic 3´,5´-guanosine monophosphate-3´,5´-adenosine monophosphate (3´,3´-cGAMP). Due to their low physiological concentrations and rapid metabolism, identification and quantification of these molecules is a highly challenging analytical task. We have developed sensitive and specific HPLC-coupled tandem mass spectrometry (LC-MS/MS) methods with the main focus on unequivocally quantification of linear and cyclic nucleotides, cyclic dinucleotides, and their respective degradation products. In this case, LC-MS/MS with inclusion of isotope-labeled internal standards and analysis of specific quantifier and several qualifier mass transitions represents the analytical method of choice. However, special care has to be taken regarding the initial sample preparation steps, the robustness of the HPLC methods, and a reliable MS/MS procedure. The established procedures can easily be adapted for the analyses of additional molecules, e.g. nucleotide analogs. It is intended in this project to further optimize the already established LC-MS/MS methods with respect to further improvement of the sample extraction procedures, establishment of specific extraction procedures for secreted signaling molecules from cellular supernatants, and biological syntheses of isotope-labeled internal standards by using recombinant enzymes. Our expertise in LC-MS/MS technology will be provided in close collaboration with many partners within the SPP 1879 as a Z unit, especially addressing the identification and quantification of bacterial nucleotide signaling molecules.