Novel analytical infrastructure
Since one major focus of our group is based on molecular intermediates and products of so-called microbial metabolites, the recently acquired mass spectrometer enables our working group to detect trace concentrations of organic molecules in otherwise complex sample matrices. Typically, the metabolite of interest vary between 50 and 2000 Da in its molecular size and have widely differing chemical and physical properties. Thus, it is extremely important to apply state-of-the-art instrumentation for detecting and identifying these structurally diverse and often unknown molecules. The Waters Synapt-G2-Si ultra-high resolution quadrupole time-of-flight mass spectrometer (Q-ToF-MS) offers high mass accuracy (<1 ppm RMS) and mass resolving power (>50.000 FWHM), as well as high scan rates of up to 30 spectra/sec. Due to the possibility to obtain exact mass data and high mass resolution in both full scan (MS1) and fragment spectra (MS2), this system is ideal to characterize and identify unknown metabolites at concentrations in the lower picogram range (on column).
Next, we introduce two possibilities how we introduce our analytes to the Q-ToF-MS; this could be either done via the Waters ACQUITY UPLC H-Class System or via the Waters MALDI source system.
Synapt G2-Si MS coupled to a Waters ACQUITY UPLC H-Class System
Liquid chromatography coupled to mass spectrometry (LC-MS) is enabling us to identify and semiquantitative determine the concentration of our target metabolite. This separation technique is based on a differential retention of organic molecules to a stationary phase. In brief, a complex analyte is injected into a stream of liquid mobile phase which is pumped at high pressure (up to 15,000 psi) through a column that is packed with a preselected stationary phase. This high-pressure application allows us to use sub-2-μm particle size columns that significantly improve our chromatographic separation, high chromatographic peak capacity as well as shorter chromatographic runs. Depending on the type and degree of interaction (i.e., interactions of alkyl chains, functional groups), the analytes are retained on the stationary phase for a certain time before eluting from the column. In a best-case scenario, a complex analyte mixture is separated into many individual compounds. Our group, most commonly applies reversed phase separation techniques, that are based on the combination of a RP column and a mobile phase gradient starting from a polar and ends at a less polar solvent mixture.
MALDI SYNAPT G2-Si System
Besides the Waters ACQUITY UPLC H-Class System we can also introduce our sample via a laser-based, high-resolution scanning techniques that enables us to spatially resolve our metabolite of interest on a µm-scale. MALDI stands for Matrix Assisted Laser Desorption Ionization and we would like to apply this technique to gain information on the distribution of a specific metabolite within potentially interesting tissue samples.