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Analysis of carbonyl-containing compounds derivatized with dinitrophenylhydrazine

Analysis of carbonyl-containing compounds derivatized with dinitrophenylhydrazine. Thi Nguyen Oregon State University. P.I.: Dr. Gary Merrill Biochemistry and Biophysics. Summer 2011. Carbonyl compounds. Menadione. Carbonyl compounds are compounds with aldehyde or ketone groups

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Analysis of carbonyl-containing compounds derivatized with dinitrophenylhydrazine

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  1. Analysis of carbonyl-containing compounds derivatized with dinitrophenylhydrazine Thi Nguyen Oregon State University P.I.: Dr. Gary Merrill Biochemistry and Biophysics Summer 2011

  2. Carbonyl compounds Menadione • Carbonyl compounds are compounds with aldehyde or ketone groups • Carbonyls are a highly reactive species • Build up of carbonyls cause carbonyl stress Pyruvate

  3. Carbonyl stress linked to diseases • Linked to modification of proteins in diseases such as atherosclerosis and Alzheimer’s disease • Atherosclerosis is slow and progressive; often leads to coronary heart disease (CHD) – the leading cause of death in America Alzheimer’s disease is the only cause of death among the top 10 in the US that cannot be prevented, slowed or cured http://www.pcna.net/national/documents/Atherosclerosis_Fact_Sheet.4.09.pdf http://www.alz.org/alzheimers_disease_facts_and_figures.asp

  4. Dinitrophenylhydrazine (DNPH) menadione-hydrazone DNPH menadione • DNPH is a good derivatizing agent for stabilizing carbonyls • Primary amine attaches to the carbonyl and forms a hydrazone

  5. Hydrazones Some hydrazones form precipitates Precipitates dissolve in different solvents depending on the hydrazone

  6. Thin layer chromatography Fig. 2. TLC analysis of DNPH, menadione, and menadione-hydrazone. Plate was immersed in NaOH, which stains the hydrazone blue. Glass silica plates Used to monitor formation of hydrazone by virtue of its blue color when exposed to base DNPH menadione menadione- hydrazone

  7. TLC mass spectrometry (TLC-MS) samples are eluted from spot on TLC plate and directly analyzed in mass spec http://www.pharmaceutical-int.com/article/tlc-mass-spectrometry.html

  8. Menadione-Hydrazone Expected mass: 352.0808 Observed mass: 351.075 Intensity, cps m/z, Da

  9. Mass spectrometry: Collision-induced dissociation (CID) M2- M1- M3- Neutral Molecules M- Parent ion Inert Gas Secondary ions

  10. CID: Menadione-Hydrazone 109.0173 secondary ion Intensity, cps diminished parent ion 351.0745 167.0107 351.0745 secondary ion m/z, Da

  11. Pyruvate-Hydrazone 267.0376 Expected mass: 268.0444 Observed mass: 267.0376 Intensity, cps m/z, Da

  12. CID: Pyruvate-Hydrazone 182.0213 secondary ion Intensity, cps secondary ion diminished parent ion 122.0252 m/z, Da

  13. Mass Spectrometry Summary The strongest candidates for a signature fragment from the DNPH-hydrazones are 182 and 122 m/z

  14. Proposed structures for secondary ions Secondary Ion 182 m/z Generic DNPH-hydrazone Secondary Ion 122 m/z

  15. Conclusion Promising signature fragments, 182 and 122 m/z, can be used in future assays to identify modeled carbonyl-compounds in cell extracts Further investigation is needed to confirm if these signature fragments apply to a wider range of DNPH-hydrazones

  16. Acknowledgements Dr. Gary Merrill Dr. Kevin Ahern Dr. Fred Stevens Dr. Jong-Tae Yang Jeff Morre AmiraBarkal OSU Mass Spectrometry Laboratory Howard Hughes Medical Institute Cripps Undergraduate Research Oregon State University

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