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

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

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

Hydrazones Some hydrazones form precipitates Precipitates dissolve in different solvents depending on the hydrazone HydrazoneFormed inPrecipitatesDissolves inColor under basic conditions Menadione1 M HCl, 23.8% EtOH YesChloroformBlue Pyruvate0.5 M HClYes95% EtOHBrown α-Ketoglutarate0.5 M HCl, 47.5% EtOH Yes95% EtOHBrown 4-Hydroxynonenal0.5 M HCl, 47.5% EtOH Non/aBrown

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

TLC mass spectrometry (TLC-MS) samples are eluted from spot on TLC plate and directly analyzed in mass spec

Menadione-Hydrazone Intensity, cps m/z, Da Expected mass: Observed mass:

Mass spectrometry: Collision-induced dissociation (CID) Parent ionInert GasSecondary ions M2-M2- M1-M1- M3-M3- Neutral Molecules M-M-

CID: Menadione-Hydrazone Intensity, cps m/z, Da diminished parent ion secondary ion

Pyruvate-Hydrazone Intensity, cps m/z, Da Expected mass: Observed mass:

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

Mass Spectrometry Summary HydrazoneParent ion (m/z) Secondary ions (m/z) menadione , pyruvate , α-ketoglutarate , , hydroxynonenal , , The strongest candidates for a signature fragment from the DNPH-hydrazones are 182 and 122 m/z

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

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

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