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Metabolite Analysis

The Metabolomics Knowledge Center is developing precise, robust methods to profile large numbers of known metabolites in complex biological samples such as serum and urine. We also develop kinetics tools for tracking the rates of enzyme-catalyzed and metal-catalyzed reactions.

Real-time measurement of of chemical and enzymatic reactions

Ever since Michaelis and Menten's 1913 work, measuring the rates of enzyme catalyzed reactions remains a central challenge in biochemistry. Often times, "continuous" assays rely on spectrophotometric or fluorescence measurements, and usually track only a single species -- a reactant or product -- of the reaction. "Discontinuous" assays -- where samples are drawn at defined times and worked up for a more detailed analysis -- can track many more chemical species, but sample workup is tedious and the time resolution of this assay mode is limited as a result.

At the Metabolomics Knowledge Center, we've sought to combine the speed and convenience of a continuous assay with the chemical resolution of a discontinuous approach using modern mass spectrometer. We set up enzymatic reactions inside our instrument's autosampler. As a result, we can sample our reactions several times per minute, tracking not just one but many reacting species at each time point. Below we show an example of simultaneously tracking substrate depletion while concomitantly tracking three distinct enzymatic products for a purified enzyme known to hydrolyze a single substrate into three distinct products.

Real time enzyme kinetic assay

Our current interests are in using this technique to monitor several competing or in-series enzymatic reactions simultaneously, and to monitor for unexpected enzyme activities in understudied proteins. As shown below, we've also recently used the same approach to perform kinetic measurements not on enzymatic reactions, but on transition-metal catalyzed reactions in organic solvents.

Transition-metal catalyzed reaction kinetics

Collaborators

Metabolite analysis to enable precision health

Clinical applications of metabolomics require robust methods with well-characterized limits of detection, sensitivities, and specificities for all analytes of interest. In conjunction with Dylan Dodd and co-workers at the Stanford School of Medicine, we are developing methods for the routine analysis of hundreds of human metabolites, both host- and endosymbiont-derived.

Collaborators

  • Dylan Dodd - Instructor, Pathology Department, Stanford University School of Medicine
  • Justin Sonnenburg - Associate Professor, Microbiology and Immunology, Stanford School of Medicine

Kidney-microbiome interactions

The role of the kidneys in removing deleterious small molecules from the human bloodstream has been appreciated for more than a century, but the precise chemical identity of the molecules that control health outcomes is not known. Recent findings have implicated intenstinal microbiota as an important determinant of the blood and urinary metabolome. In collaboration with nephrologist Tim Meyer and his team at the Stanford school of medicine, we are probing the link between kidney function and metabolites that are derived from intestinal microbiota.

Collaborators

  • Tim Meyer - Professor, Nephrology Department, Stanford University School of Medicine
  • Tammy Sirich
  • Robert Mair