Untargeted NMR Metabolomics of Glioblastoma
About the project
Primary glioblastoma cells were received from the Institute of Clinical Medicine, University of Oslo (UiO). The cell samples received fall 2016 were grown by MD and fellow Erlend Skaga, samples received from December 2016 to April 2017 were grown by fellow Marit Christensen, and samples received August and September 2017 were grown by research technician Maria Ewa Walewska.
In the preliminary NMR studies a variety of cell samples were analyzed: five different cell lines, four different chemotherapies, in addition to control (untreated) samples, and five different samples cell numbers.
For the final NMR procedure, five different cell lines, two different chemotherapies, in addition to control (untreated) samples were analyzed. All samples contained the same amount of cells, three million.
Samples were kept at 7 °C in the SampleCase prior to and after analysis. Temperature inside the probe was kept at 300 K. Samples were given 15 minutes inside the probe before start of analysis for temperature equilibration.
One dimensional proton NMR (1D 1H NMR) was done with excitation sculpting with gradient pulses for water suppression. Pulse program zesgp. Number of scans was 4096 and number of dummy scans was 4.
Two different two dimensional proton NMR spectra were acquired. J resolved spectrum (JRES) with pulse program jresgpprqf. The spectra were acquired with Non Uniform Sampling (NUS), and had a NUS percent at 25% (40 NUS points). Total Correlation Spectroscopy (TOCSY) spectra were acquired with the pulse program dipsi2esgpph. The spectra were acquired with NUS and had a NUS percent at 25 % (128 NUS points).
Statistical Analysis: Principle Component Analysis
Principle component analysis (PCA) was done with the program R (version R x64 3.1.2 RC) with the help of PhD Daniel Sachse. Only one dimensional proton NMR spectra were used for the analysis. The data was processed in TopSpin (phase correction, referencing to the TSP peak) and R (removal of unwanted peaks and Normalization) prior to the execution of the PCA.
The AVIIIHD800 Bruker nmr instrument at the University of Oslo NMR Center is used in these studies.