期刊
ANALYTICAL CHEMISTRY
卷 79, 期 17, 页码 6629-6640出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac070843+
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资金
- NHLBI NIH HHS [P01 HL57278, P01 HL057278, P01 HL057278-10] Funding Source: Medline
- NIDDK NIH HHS [P30 DK056341-06, P30 DK056341, P30 DK056341-07] Funding Source: Medline
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.
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