Metabolomics provides a comprehensive quantitative analysis of small molecules and cellular pathways and a powerful instrument to monitor metabolic perturbations occurring during physiological processes or pathological changes. While just years ago, technical limitations prevented more than one or a few molecules from being explored at one time, the omics revolution facilitated the simultaneous survey of hundreds or thousands of molecules that belong to multiple biological pathways.
Of several approaches used to quantitate thousands of molecules at the same time, separation methods coupled with mass spectrometry have gained widespread use. Nevertheless, while mass spectrometry provides a precise measurement of the molecular mass and can reliably differentiate molecules, it is also accompanied by shortcomings.
“A major challenge is being able to quantify these single molecules,” says Jiri Adamec, Ph.D., lead scientist and director of the proteomics/ metabolomics core facilities at Bindley Bioscience Center, Purdue University. Dr. Adamec will be a speaker at Select Biosciences’ “Advances in Metabolic Profiling” meeting to be held in Barcelona in November.
“In terms of quantification, mass spectrometry is not reporting the overall concentration of the compound but, rather, the ionization efficiency,” he explains. This means that a molecule that does not ionize well might be difficult to measure, despite its high concentrations, while another molecule present at lower concentrations can generate huge signals and be overrepresented during measurements, if it ionizes well.
Research in Dr. Adamec’s laboratory is focusing on modifying molecules with well-defined moieties, for example, the ones containing permanent positive charges that increase ionization efficiency.
A second important challenge for quantification is that ionization efficiency does not depend only on concentration, but is also shaped by surrounding compounds that may suppress ionization, a phenomenon known as the ion-suppression effect. Investigators in Dr. Adamec’s lab solved this problem by differentially isotope labeling two samples and subsequently analyzing them in one run.
With this approach, the relative abundance of the labeled molecules can be measured in a mixture, or when the samples are mixed with a known amount of isotopically labeled internal standards, absolute quantifications can be performed as well. Based on this principle, Dr. Adamec’s lab used in vitro aniline derivatization to quantitate metabolites in the energy and carbon metabolic pathways, and were able to analyze over 35 compounds, including carboxylic acids, phosphorylated sugars, nucleotides, and intermediates belonging to several cellular metabolic pathways.
A current effort in Dr. Adamec’s lab is a targeted analysis focusing on vitamin D derivatization that proposes to develop specific reagents to analyze low concentrations of this vitamin and related metabolites in biological fluids and tissues. This tool would address the low sensitivity, selectivity, and reliability of currently available approaches.