We have established a targeted liquid chromatography-mass spectrometry-based metabolomics platform in collaboration with the Broad Institute. In its current form, the platform analyzes ~380 sugars, sugar phosphates, nucleotides, amino acids, organic acids, and lipids in a targeted manner. Our platform is designed on the principle that a metabolite can be identified by its parent mass (MS) and dominant daughter mass (MS/MS) on a high resolution mass spectrometer in combination with its retention time on an appropriate HPLC column. While none of these three parameters is individually sufficient to uniquely identify a metabolite in a biological fluid, the three (in combination) provide a “tag” that marks the metabolite to permit selective reaction monitoring and quantitation.
When we established this system, we purchased ~250 compounds, created standards, and empirically determined elution properties on each of three different columns, their parent masses (MS), and daughter (fragmentation MS/MS) masses. These standards also enable us to calibrate our instrument on a daily basis. We then ensured that these parameters were valid in plasma or in cell culture media where matrix effects often necessitate further modifications. These measures provide the parameters needed for multiple reaction monitoring of these metabolites in more complex fluids.
We have optimized multiple parameters to improve both the throughput and the sample volume necessary for the analysis, which is based on an Applied Biosystems 4000 QTRAP triple quadrupole mass spectrometer. The first quadrupole (Q1) allows passage of only parent masses within a narrow mass window. Following collision-induced dissociation in the second quadrupole (Q2), only selected daughter ions are monitored in the third quadrupole (Q3) where peak areas are quantitated. In addition to analyzing “endogenous” metabolites, we also routinely monitor human plasma levels of 24 drugs commonly used to treat cardiovascular and metabolic diseases. The present platform can acquire the data from 200 mL of plasma.
Please contact Dr. Robert Gerszten by e-mail (firstname.lastname@example.org).
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