University of Colorado Boulder & Institute for Arctic and Alpine Research (INSTAAR), Boulder, CO, USA.
Thermo Fisher Scientific GmbH, Bremen, Germany.
Nat Protoc. 2024 Aug;19(8):2435-2466. doi: 10.1038/s41596-024-00981-5. Epub 2024 Apr 23.
Stable isotopes of carbon, hydrogen, nitrogen, oxygen and sulfur are widespread in nature. Nevertheless, their relative abundance is not the same everywhere. This is due to kinetic isotope effects in enzymes and other physical principles such as equilibrium thermodynamics. Variations in isotope ratios offer unique insights into environmental pollution, trophic relationships in ecology, metabolic disorders and Earth history including climate history. Although classical isotope ratio mass spectrometry (IRMS) techniques still struggle to access intramolecular information like site-specific isotope abundance, electrospray ionization-Orbitrap mass spectrometry can be used to achieve precise and accurate intramolecular quantification of isotopically substituted molecules ('isotopocules'). This protocol describes two procedures. In the first one, we provide a step-by-step beginner's guide for performing multi-elemental, intramolecular and site-specific stable isotope analysis in unlabeled polar solutes by direct infusion. Using a widely available calibration solution, isotopocules of trifluoroacetic acid and immonium ions from the model peptide MRFA are quantified. In the second approach, nitrate is used as a simple model for a flow injection routine that enables access to a diverse range of naturally occurring isotopic signatures in inorganic oxyanions. Each procedure takes 2-3 h to complete and requires expertise only in general mass spectrometry. The workflows use optimized Orbitrap IRMS data-extraction and -processing software and are transferable to various analytes amenable to soft ionization, including metabolites, peptides, drugs and environmental pollutants. Optimized mass spectrometry systems will enable intramolecular isotope research in many areas of biology.
碳、氢、氮、氧和硫的稳定同位素在自然界中广泛存在。然而，它们的相对丰度并非处处相同。这是由于酶和其他物理原理（如平衡热力学）中的动力学同位素效应所致。同位素比值的变化为环境污染、生态学中的营养关系、代谢紊乱以及包括气候历史在内的地球历史提供了独特的见解。尽管经典的同位素比质谱（IRMS）技术仍然难以获取像位点特异性同位素丰度这样的分子内信息，但电喷雾电离-轨道阱质谱可以用于对同位素取代分子（“同位素簇”）进行精确和准确的分子内定量。本协议描述了两种程序。在第一种程序中，我们提供了一个逐步的初学者指南，用于通过直接注入对未标记的极性溶质进行多元素、分子内和位点特异性稳定同位素分析。使用广泛可用的校准溶液，对模型肽 MRFA 的三氟乙酸和亚铵离子的同位素簇进行定量。在第二种方法中，硝酸盐被用作一种简单的模型，用于流动注射常规，从而能够获得各种天然存在的无机含氧阴离子的同位素特征。每个程序需要 2-3 小时才能完成，并且仅需要一般质谱方面的专业知识。这些工作流程使用优化的轨道阱 IRMS 数据提取和处理软件，并可转移到各种易于软电离的分析物，包括代谢物、肽、药物和环境污染物。优化的质谱系统将使生物学许多领域的分子内同位素研究成为可能。
