A global (or untargeted approach) to metabolite profiling involves monitoring as many
metabolites in the entire metabolome as possible. LC/MS is often used for this task. A complimentary approach is GCxGC/MS. Whereas LC/MS is able to monitor
hundreds of peaks in a single run, the GCxGC/MS has a peak capacity in the thousands.
Below, is an example of a plant leaf tissue extract assayed on the
GCxGC/MS. Over 4,000 compounds were detected.
Many projects involve the quantitation of predetermined analytes in a complex biological matrix. This type of targeted analysis requires that analytical methods not only be developed, but also validated for specificity, linearity, range, precision, detection limit, quantitation limit, accuracy, robustness, and solution stability. In complex biological mixtures, internal standards should be utilized to correct for the loss of analyte during sample preparation or introduction into the instrument. With mass spectrometry, stable isotopes provide the greatest accuracy for quantitation.
Amino Acids have been successfully analyzed using GC/MS. The samples are
derivatized to produce a TBDMS (tert-butyldimethylsilyl) derivative. This label will
derivatize both the carboxyl and amino group of the amino acid, as well as any hydroxyl,
carboxyl, thiol or primary and secondary amine that may appear in the side chain. These
derivatives are 10,000 times more stable than traditional TMS ethers. The separation can
be conducted in 30 minutes. The detection limit is low picograms injected on-column.
Useful internal standards are 2-aminobutyric acid and beta-alanine.
Sugars (monosaccharides) represent a challenging class of compounds to analyze. We
have been successful in analyzing this class using GCxGC/MS. The samples are
derivatized, by performing a methoxymation to prevent ring formation followed by a
trimethylsilylation with MSTFA.
A method for general profiling of phospholipids using the LC/MS TOF or Thermo Orbitrap has been developed. The chromatography is
designed to separate different phospholipid classes based on head group chemistry. The protocol was developed to analyze a typical lipid extract
in negative ESI mode. Phosphatidylserine (POPS), phosphatidic acid (POPA), phosphatidylglycerol (POPG), phosphatidylethanolamine (POPE),
and ceramides can be analyzed using this protocol. Phosphatidylcholines and sphingolipids can be analyzed using positive ESI mode.
Metabolite Profiling Facility (MPF)
Bindley Bioscience Center
1203 W. State St.
West Lafayette, IN 47907-2010
- (765) 494-6282
Bruce Cooper, Ph.D.
- (765) 494-6282
Amber Hopf Jannasch
- (765) 496-1756