A Facile and Sensitive Method for Quantification of Cyclic Nucleotide Monophosphates in Mammalian Organs: Basal Levels of Eight cNMPs and Identification of 2',3'-cIMP
Abstract
:1. Introduction
2. Results and Discussion
2.1. Optimization of LC-MS/MS Analytical Method
Parameters | 2',3'-cAMP | 3',5'-cAMP | 2',3'-cCMP | 3',5'-cCMP | 2',3'-cGMP | 3',5'-cGMP | 3',5'-cIMP | 8-79Br-cAMP | 8-81Br-cAMP |
---|---|---|---|---|---|---|---|---|---|
[M + H]+ (m/z) | 330.1 | 330.1 | 306.1 | 306.1 | 346.1 | 346.1 | 331.0 | 408.0 | 410.0 |
Production (m/z) | 136.1 | 136.1 | 112.1 | 112.1 | 152.1 | 152.1 | 137.1 | 214.0 | 216.0 |
Ret. time (min) | 6.8 | 12.9 | 1.6 | 4.0 | 15.6 | 24.3 | 24.1 | 26.3 | 26.3 |
LOD (fmol) | 273 | 153 | 171 | 455 | 94 | 487 | 219 | NA | NA |
LOQ (fmol) | 910 | 510 | 570 | 1517 | 313 | 1623 | 730 | NA | NA |
2.2. LC-MS/MS Method Calibration and Limits of Detection
2.3. Optimization of Extraction Method
2.4. Method Validation and Reproducibility Tests
Sample | 2',3'-cAMP | 3',5'-cAMP | 2',3'-cCMP | 3',5'-cCMP | 2',3'-cGMP | 3',5'-cGMP | 3',5'-cIMP |
---|---|---|---|---|---|---|---|
Portion 1 | 9.9 ± 0.2 | 88.7 ± 12.3 | 1.2 ± 0.9 | 0.9 ± 0.7 | 2.5 ± 0.01 | 10.1 ± 2.3 | 2.6 ± 0.9 |
Portion 2 | 9.6 ± 0.3 | 92.3 ± 2.9 | ND | 0.6 ± 0.4 | 1.8 | 8.7 ± 0.3 | 2.3 ± 0.3 |
Portion 3 | 9.7 ± 0.2 | 87.9 ± 4.8 | 1.3 | 0.6 ± 0.4 | 1.8 ± 0.5 | 8.3 ± 0.7 | 3.1 ± 0.3 |
Average | 9.7 ± 0.2 | 89.6 ± 2.3 | 1.3 ± 0.1 | 0.7 ± 0.1 | 2.0 ± 0.4 | 9.0 ± 0.9 | 2.7 ± 0.4 |
Inter-run Precision | 2.1 | 2.6 | 7.7 | 14.3 | 20.0 | 10.0 | 14.8 |
2.5. Applications
2.6. Versatility of the Method
Replicate | 2',3'-cAMP | 3',5'-cAMP | 2',3'-cCMP | 3',5'-cCMP | 2',3'-cGMP | 3',5'-cGMP | 2',3'-cIMP | 3',5'-cIMP |
---|---|---|---|---|---|---|---|---|
1 | 5.1 ± 0.3 | 4.8 ± 0.1 | 1.3 ± 0.1 | N/D | 2.1 ± 0.1 | N/D | N/D | N/D |
2 | N/D | 167.1 ± 35.0 | N/D | N/D | 32.0 ± 2.3 | 17.2 ± 0.3 | N/D | N/D |
3 | N/D | 99.8 ± 0.2 | 1.8 | N/D | 19.9 ± 0.2 | 8.0 ± 3.7 | N/D | N/D |
Average | 5.1 | 90.6 ± 81.6 | 1.6 ± 0.3 | N/D | 18.0 ± 15.0 | 12.6 ± 4.6 | N/D | N/D |
2.7. Discussion
3. Experimental
3.1. Materials
3.2. Calibration Curves
3.3. NIH-3T3 Cell Growth
3.4. cNMP Extraction from Rat Organs
3.5. cNMP Extraction from NIH-3T3 Cell Line
3.6. LC-MS/MS Sample Preparation
3.7. LC-MS/MS Optimized Conditions
3.8. Statistical Analysis
3.9. Method Validation Test
3.10. Method Reproducibility Test
3.11. Synthesis of 2',3'-cIMP
4. Conclusions
Supplementary Files
Supplementary File 1Acknowledgments
Author Contributions
Conflicts of Interest
References
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Jia, X.; Fontaine, B.M.; Strobel, F.; Weinert, E.E. A Facile and Sensitive Method for Quantification of Cyclic Nucleotide Monophosphates in Mammalian Organs: Basal Levels of Eight cNMPs and Identification of 2',3'-cIMP. Biomolecules 2014, 4, 1070-1092. https://doi.org/10.3390/biom4041070
Jia X, Fontaine BM, Strobel F, Weinert EE. A Facile and Sensitive Method for Quantification of Cyclic Nucleotide Monophosphates in Mammalian Organs: Basal Levels of Eight cNMPs and Identification of 2',3'-cIMP. Biomolecules. 2014; 4(4):1070-1092. https://doi.org/10.3390/biom4041070
Chicago/Turabian StyleJia, Xin, Benjamin M. Fontaine, Fred Strobel, and Emily E. Weinert. 2014. "A Facile and Sensitive Method for Quantification of Cyclic Nucleotide Monophosphates in Mammalian Organs: Basal Levels of Eight cNMPs and Identification of 2',3'-cIMP" Biomolecules 4, no. 4: 1070-1092. https://doi.org/10.3390/biom4041070