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A deoxynucleotide derivatization methodology for improving LC-ESI-MS detection.
Jimmy Flarakos1, Wennan Xiong, James Glick
1Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA.
Analytical Chemistry
|April 15, 2005
Summary
We developed a novel derivatization method for analyzing deoxyguanosine monophosphate (dGMP) adducts. This technique enhances signal intensity in positive ion mode mass spectrometry, improving detection of DNA nucleotide adducts.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Mass Spectrometry
Background:
- DNA nucleotide adducts are crucial biomarkers for exposure to carcinogens.
- Traditional detection methods in negative ion mode suffer from poor chromatographic separation and low MS sensitivity.
- Novel derivatization strategies are needed to improve the detection of these adducts.
Purpose of the Study:
- To develop and validate a novel derivatization method for deoxyguanosine monophosphate (dGMP) adducts.
- To enhance signal intensity and improve detection limits using positive ion mode electrospray ionization mass spectrometry (ESI-MS).
- To apply the method for sensitive detection of DNA adducts in biological samples.
Main Methods:
- Synthesized deoxyguanosine nucleotide phosphoramidates and phosphomonoesters using peptide coupling reagents.
- Characterized derivatives using MS/MS and determined conversion yields via DAD-UV.
- Evaluated derivatives' ionization efficiencies, fragmentation, and chromatographic properties by LC/ESI-MS/MS.
- Utilized a model system with N-acetoxy-2-acetylaminofluorene (AAAF) to create and derivatize a dGMP adduct (dGpAAF).
Main Results:
- Hydrophobic dGMP derivatives exhibited increased ionization efficiency and improved chromatographic peak shape.
- Hexamethyleneimine proved to be the optimal derivatizing agent, yielding a 3-4 fold signal enhancement for dGp.
- Detection limits for derivatized dGp (dGphex) and dGpAAF (dGpAAFhex) were significantly improved (10- and 3-fold higher S/N, respectively).
- Demonstrated practical applicability by detecting AAAF-induced DNA adducts in calf thymus DNA at the low-femtomole level.
Conclusions:
- Developed a simple and effective derivatization method for enhanced detection of dGMP and its adducts.
- The method enables sensitive analysis of DNA adducts in positive ion mode ESI-MS.
- This technique offers a significant advancement for biomarker discovery and toxicological studies.