Bindley Bioscience Center

Core Capabilities

Metabolite Profiling

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.

Quantitation

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

Amino Acids 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.

Carbohydrate Analysis

Carbohydrate Analysis 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.

Phospholipid Analysis

Carbohydrate Analysis 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.