Shotgun metabolomics approach for the analysis of negatively charged water-soluble cellular metabolites from mouse heart tissue
Shotgun metabolomics approach for the analysis of negatively charged water-soluble cellular metabolites from mouse heart tissue
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DOI：
10.1021/ac070843
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发表时间：
2007-09-01
影响因子：
7.4
通讯作者：
Gross, Richard W.
中科院分区：
文献类型：
--
作者：
Sun, Gang;Yang, Kui;Gross, Richard W.
建立了一种基于MALDI-TOF/TOF质谱的鸟枪代谢组学方法，用于快速分析带负电荷的水溶性细胞代谢物。通过使用中性有机溶剂来抑制内源酶活性（即，甲醇/氯仿/H2O提取），结合具有最小背景噪声的基质（9-氨基吖啶），开发了一组多重条件，其允许鉴定对应于来自小鼠心脏提取物的带负电荷的代谢物的285个峰。代谢物峰的鉴别基于质量准确度，并通过串联质谱法对90个已鉴别代谢物峰进行了确认。通过多重电离条件，新的代谢物组可以被电离，并且可以实现用于随后的串联质谱询问的紧密相邻峰的“光谱分离”。此外，在许多情况下，通过串联质谱法通过鉴定诊断碎片离子（例如，区分ATP和dGTP）。这种方法的高灵敏度有助于检测丰度极低的代谢物，包括重要的信号传导代谢物，如IP 3、cAMP和cGMP。总的来说，这些结果确定了一个多路复用MALDI-TOF/TOF MS方法分析哺乳动物组织中带负电荷的代谢物。
A shotgun metabolomics approach using MALDI-TOF/TOF mass spectrometry was developed for the rapid analysis of negatively charged water-soluble cellular metabolites. Through the use of neutral organic solvents to inactivate endogenous enzyme activities (i.e., methanol/chloroform/H2O extraction), in conjunction with a matrix having minimal background noise (9-amnioacridine), a set of multiplexed conditions was developed that allowed identification of 285 peaks corresponding to negatively charged metabolites from mouse heart extracts. Identification of metabolite peaks was based on mass accuracy and was confirmed by tandem mass spectrometry for 90 of the identified metabolite peaks. Through multiplexing ionization conditions, new suites of metabolites could be ionized and "spectrometric isolation" of closely neighboring peaks for subsequent tandem mass spectrometric interrogation could be achieved. Moreover, assignments of ions from isomeric metabolites and quantitation of their relative abundance was achieved in many cases through tandem mass spectrometry by identification of diagnostic fragmentation ions (e.g., discrimination of ATP from dGTP). The high sensitivity of this approach facilitated the detection of extremely low abundance metabolites including important signaling metabolites such as IP3, cAMP, and cGMP. Collectively, these results identify a multiplexed MALDI-TOF/TOF MS approach for analysis of negatively charged metabolites in mammalian tissues.