Technology Development

Over the last few decades, mass spectrometry has developed into the technique of choice for analysis of proteins and metabolites.  The Evans Lab is continually working on development of methods to expand the depth of information the can be gleaned using chromatographic separations and mass spectrometry.  Some examples of ongoing work in the lab include:

• Differential mobility separation of isomeric/isobaric species
• Multidimensional chromatographic separations for increased metabolomic coverage
• Sample preparation methods for quantitative phosphoproteomic analysis
• Automated data analysis tools for untargeted metabolomics analysis

Inositol Polyphosphates

Inositol (poly)phosphates (IPs) are important cellular signaling molecules in eukaryotic organisms having diverse roles from calcium signaling pathways to telomere maintenance.  There are theoretically 63 different isomers of IPs possible, not considering the pyrophosphates.  So far, over 30 IP isomers have been found in eukaryotic cells.  In spite of the important roles that IPs play in cellular function and the many possible isomers known to exist, robust methods for detection and quantitation of these very low abundance metabolites exist.  In collaboration with the Umen Lab and the Allen Lab at the Danforth Center, the Evans Lab is developing sensitive and selective mass spectrometric methods that use differential mobility spectrometry in conjunction with chromatography to separate and quantify IP isomers.

Natural Products

Plants produce a vast array of specialized metabolites, or natural products that communicate with other organisms, defend against pathogens and herbivores, attract symbionts and deter pests. These metabolites are produced by both wild and cultivated species, including important crops.  Plant natural products are important for plant and crop defense and can also be used as agricultural chemicals, fine chemicals and medicines.  Understanding the genes responsible for production of plant specialized metabolites will be useful for maximal exploitation of these metabolites for crop protection and other human uses. However, linking the chemical phenotype (chemotype) of a plant to its underlying genotype is arduous and slow. The conventional process begins with purifying chemical compounds from the plant, determining their structures, and using reverse genetics approaches to identify the relevant biosynthetic genes.  Finally, in vivo and in vitro studies are required to study gene and pathway function in the plant.  There are serval hurdles in applying this workflow.  First, the chemical compound must be produced constitutively or at least under conventional laboratory conditions.  Second, the compound(s) of interest must be flagged as interesting using a biological or chemical assay. Third the compound(s) must be de-replicated—in other words it must be determined if the biological activity or chemical signature comes from a compound that has already been characterized or if it comes from a novel compound, a process that has historically been very time consuming.  Finally, validation requires genes coding for putative biosynthetic enzymes to be disrupted and/or cloned and heterologously expressed.  The Evans Lab is actively working on methods using high-resolution accurate-mass mass spectrometry for quick dereplication of natural product extracts as well as methods for accurate structural classification and comparative analyses of plant metabolomes.