Isotope Dilution - Thermal Ionisation Mass Spectrometric ...
Natural Abundance and Labeled Compounds · Silverstein, Robert M., Francis X. Webster, and David J....
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Quantification of 13 C Labeled Compounds Within a Mixture of Natural Abundance and Labeled Compounds Keiko Cooley 1 , Chaevien Clendinen 2 , Arthur S. Edison 2 1 Department of Biochemistry, School of Chemistry, Claflin University, Orangeburg, SC 29115 2 Department of Biochemistry, University of Florida, Gainesville, FL 32608 INTRODUCTION EXPERIMENTAL DATA RESULTS METHODS INSTRUMENTATION REFERENCES ACKNOWLEDGEMENTS I would like to thank the National Science Foundation, the National Magnetic Field Laboratory, the University of Florida, the members of the Edison Laboratory, Chaevien Clendinen and Greg Stupp. I would also like to thank Jim Rocca of the University of Florida. My home institution of Claflin University, Angela Peters, Muthukrishna Raja, and Arezue F. Boroujerdi. Sample preparation required the following instrumentation: 2, 10, 100, and 1000 µL pipettes, a 40 µL syringe, an analytical balance, and a shaker. Sample data was collected on an Agilent VNMRS-600 MHz spectrometer that is 14.1 Tesla Magnex magnet equipped with a 5-mm cryogenically cooled radio-frequency probe. The spectrometer operates with the system of VNMR-J 2.3 software. Both the 1D Proton and 1D HSQC spectra data were processed in MestReNova. In MATLAB, Principal Component Analysis was completed. Two groups of four identical replicates each were made using twelve unlabeled (natural abundance) carbon compounds and six 13 C labeled compounds. The concentration of each unlabeled compound was randomly generated on the range of 0.5 mM to 4 mM. The six 13 C labeled concentrations were constant within both groups. The NMR spectra were collected using an Agilent VNMRS-600 MHz spectrometer. The data were then preprocessed using MestReNova and MATLAB. In MestReNova the FID was zero filled and a window function was applied. The FID was then Fourier transformed. After, spectrum was then phased and baseline corrected. In MATLAB, Principal Component Analysis (PCA) was completed. After analysis of the data it was determined that the concentration of glucose in the 1D proton spectrum did not match the concentration of glucose in the 1D HSQC. This could be because of errors in pipetting the sample or the addition of too much TSP. It was seen that in the performance of PCA that the groups were segregated successfully. It shows that the software successfully recognized a patterns within the data and output those patterns to show their similarities and differences. This project was completed to explore the possibility of quantifying 13 C labeled compounds within in a mixture and to see how efficiently it could be done. One-dimensional proton spectra were collected and 1D Heteronuclear Single Quantum Coherence (HSQC) was also collected. Unlike the 1D proton spectra, that shows all protons, an HSQC only shows protons that are connected to the 13 C labeled compounds. The goal was to determine if 13 C labeled compounds could be quantified within a mixture of labeled and unlabeled compounds with the use of Nuclear Magnetic Resonance (NMR) Spectroscopy and analysis of spectra. Stock solutions were made using 18 different compounds. Six of the 18 compounds were 13 C labeled. There were two groups made with a consistent concentration of 13 C labeled isotopes. Four replicates for each group were made. Each replicate was analyzed using NMR spectroscopy. One dimensional proton and HSQC spectra were collected and analyze. The focus of this poster will be 13 C Glucose. 1. Creighton, Thomas E.. "Chemical Properties of Polypeptides." Proteins: structures and molecular properties. 2. ed. New York, N.Y.: W.H. Freeman, 1996. 2-4. Print. 2. Silverstein, Robert M., Francis X. Webster, and David J. Kiemle. "Proton NMR Spectrometry." Spectrometric identification of organic compounds. 7th ed. Hoboken, NJ: John Wiley & Sons, 2005. 127-203. Print. FUTURE GOALS To quantity the 1D proton spectra in its entirety. Plans also include the quantifying of the 1D HSQC spectra. Analysis aims include the analysis of ratios between the 1D proton and 1D HSQC to compare the concentration of the 13 C satellites to the 13 C peaks. The parameters of how the 1D proton spectra were collected include the following: temperature at 25°C, with a pulse of 45°, a relaxation time of 2 seconds and an acquisition time of 3 seconds. There were a total of 32 scans acquired. The parameters of how the 1D HSQC proton spectra were collected include the following: temperature of 25°C, with a pulse of 90°, a relaxation time of 1.5 seconds and an acquisition time of 0.15 seconds. There was a total of 128 scan acquired. a b Figure 1: Instrumentation used for samples. (a) NMR spectrometer that was used for the data collection. Figure (b) 40 microliter syringe used to place the sample in the capillary tubes. a b c Figure 2: Collected and Processed Experimental Data. (a) Free Induction Decay (FID) of a 1D proton spectrum. It is the data that is collected from the scans during acquisition of an NMR experiment. (b) A 1D Proton spectrum after preprocessing is complete. (c) A 1D HSQC after preprocessing is complete. Figure 3: Analysis of Preprocessed Spectra (a) The red spectrum is the collected HSQC. The green spectrum is a downloaded Glucose spectrum for reference. (b) 1D proton spectrum. The blue peaks are natural abundance protons. At approximately 5.2 ppm the smaller adjacent peaks are 13 C satellites that have been split. (c) PCA scores plot showing the variance and similarity between the two groups. (d) A loadings plot showing the corresponding contributions each compound makes. a. b. c. d.
Transcript of Natural Abundance and Labeled Compounds · Silverstein, Robert M., Francis X. Webster, and David J....
Quantification of 13C Labeled Compounds Within a Mixture of Natural Abundance and Labeled Compounds
Keiko Cooley1, Chaevien Clendinen2 , Arthur S. Edison2
1Department of Biochemistry, School of Chemistry, Claflin University, Orangeburg, SC 29115 2Department of Biochemistry, University of Florida, Gainesville, FL 32608
INTRODUCTION EXPERIMENTAL DATA
RESULTS
METHODS INSTRUMENTATION
REFERENCES ACKNOWLEDGEMENTS I would like to thank the National Science Foundation, the National Magnetic Field Laboratory, the University of Florida, the members of the Edison Laboratory, Chaevien Clendinen and Greg Stupp. I would also like to thank Jim Rocca of the University of Florida. My home institution of Claflin University, Angela Peters, Muthukrishna Raja, and Arezue F. Boroujerdi.
Sample preparation required the following instrumentation: 2, 10, 100, and 1000 µL pipettes, a 40 µL syringe, an analytical balance, and a shaker. Sample data was collected on an Agilent VNMRS-600 MHz spectrometer that is 14.1 Tesla Magnex magnet equipped with a 5-mm cryogenically cooled radio-frequency probe. The spectrometer operates with the system of VNMR-J 2.3 software. Both the 1D Proton and 1D HSQC spectra data were processed in MestReNova. In MATLAB, Principal Component Analysis was completed.
Two groups of four identical replicates each were made using twelve unlabeled (natural abundance) carbon compounds and six 13C labeled compounds. The concentration of each unlabeled compound was randomly generated on the range of 0.5 mM to 4 mM. The six 13C labeled concentrations were constant within both groups. The NMR spectra were collected using an Agilent VNMRS-600 MHz spectrometer. The data were then preprocessed using MestReNova and MATLAB. In MestReNova the FID was zero filled and a window function was applied. The FID was then Fourier transformed. After, spectrum was then phased and baseline corrected. In MATLAB, Principal Component Analysis (PCA) was completed.
After analysis of the data it was determined that the concentration of glucose in the 1D proton spectrum did not match the concentration of glucose in the 1D HSQC. This could be because of errors in pipetting the sample or the addition of too much TSP. It was seen that in the performance of PCA that the groups were segregated successfully. It shows that the software successfully recognized a patterns within the data and output those patterns to show their similarities and differences.
This project was completed to explore the possibility of quantifying 13C labeled compounds within in a mixture and to see how efficiently it could be done. One-dimensional proton spectra were collected and 1D Heteronuclear Single Quantum Coherence (HSQC) was also collected. Unlike the 1D proton spectra, that shows all protons, an HSQC only shows protons that are connected to the 13C labeled compounds.
The goal was to determine if 13C labeled compounds could be quantified within a mixture of labeled and unlabeled compounds with the use of Nuclear Magnetic Resonance (NMR) Spectroscopy and analysis of spectra.
Stock solutions were made using 18 different compounds. Six of the 18 compounds were 13C labeled. There were two groups made with a consistent concentration of 13C labeled isotopes. Four replicates for each group were made. Each replicate was analyzed using NMR spectroscopy. One dimensional proton and HSQC spectra were collected and analyze. The focus of this poster will be 13C Glucose.
1. Creighton, Thomas E.. "Chemical Properties of Polypeptides." Proteins: structures and molecular properties. 2. ed. New York, N.Y.: W.H. Freeman, 1996. 2-4. Print.
2. Silverstein, Robert M., Francis X. Webster, and David J. Kiemle. "Proton NMR Spectrometry." Spectrometric identification of organic compounds. 7th ed. Hoboken, NJ: John Wiley & Sons, 2005. 127-203. Print.
FUTURE GOALS To quantity the 1D proton spectra in its
entirety. Plans also include the quantifying of the 1D HSQC spectra. Analysis aims include the analysis of ratios between the 1D proton and 1D HSQC to compare the concentration of the 13C satellites to the 13C peaks.
The parameters of how the 1D proton spectra were collected include the following: temperature at 25°C, with a pulse of 45°, a relaxation time of 2 seconds and an acquisition time of 3 seconds. There were a total of 32 scans acquired. The parameters of how the 1D HSQC proton spectra were collected include the following: temperature of 25°C, with a pulse of 90°, a relaxation time of 1.5 seconds and an acquisition time of 0.15 seconds. There was a total of 128 scan acquired.
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b
Figure 1: Instrumentation used for samples. (a) NMR spectrometer that was used for the data collection. Figure (b) 40 microliter syringe used to place the sample in the capillary tubes.
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Figure 2: Collected and Processed Experimental Data. (a) Free Induction Decay (FID) of a 1D proton spectrum. It is the data that is collected from the scans during acquisition of an NMR experiment.(b) A 1D Proton spectrum after preprocessing is complete. (c) A 1D HSQC after preprocessing is complete.
Figure 3: Analysis of Preprocessed Spectra (a) The red spectrum is the collected HSQC. The green spectrum is a downloaded Glucose spectrum for reference. (b) 1D proton spectrum. The blue peaks are natural abundance protons. At approximately 5.2 ppm the smaller adjacent peaks are 13C satellites that have been split. (c) PCA scores plot showing the variance and similarity between the two groups. (d) A loadings plot showing the corresponding contributions each compound makes.
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