Shotgun metabolomics approach for the analysis of negatively charged water-soluble cellular metabolites from mouse heart tissue.
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.
Sun
Gang
G
Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Medicine, Washington University School of Medicine, St Louis, MO 63110, USA.
Yang
Kui
K
Zhao
Zhongdan
Z
Guan
Shaoping
S
Han
Xianlin
X
Gross
Richard W
RW
D000818:Animals; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D007536:Isomerism; D051379:Mice; D008810:Mice, Inbred C57BL; D015394:Molecular Structure; D032383:Myocytes, Cardiac / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014020:Tissue Extracts / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D014867:Water / Q000737:chemistry*
D007477:Ions; D014020:Tissue Extracts; D014867:Water; D000242:Cyclic AMP