Analysis of carbonyl-containing compounds

derivatized with dinitrophenylhydrazine

Thi NguyenOregon State University

P.I.: Dr. Gary MerrillBiochemistry 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

HO

O

O

O

O

Carbonyl stress linked to diseasesLinked to modification of proteins in

diseases such as atherosclerosis and Alzheimer’s disease

http://www.alz.org/alzheimers_disease_facts_and_figures.asphttp://www.pcna.net/national/documents/Atherosclerosis_Fact_Sheet.4.09.pdf

Atherosclerosis is slow and progressive; often leads to coronary heart disease (CHD) – the leading cause of death in AmericaAlzheimer’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 carbonylsPrimary amine attaches to the carbonyl and forms a hydrazone

O

N

NH

NO2

NO2

menadione-hydrazone

H2N

NH

NO2

NO2

O

O

menadione DNPHDNPHmenadione

menadione-hydrazone

HydrazonesSome hydrazones form precipitatesPrecipitates dissolve in different solvents depending on the hydrazone

Hydrazone Formed in

Precipitates

Dissolves in

Color under basic conditions

Menadione 1 M HCl, 23.8% EtOH

Yes Chloroform

Blue

Pyruvate 0.5 M HCl Yes 95% EtOH Brown

α-Ketoglutarate 0.5 M HCl, 47.5% EtOH

Yes 95% EtOH Brown

4-Hydroxynonenal

0.5 M HCl,47.5% EtOH

No n/a Brown

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

http://www.pharmaceutical-int.com/article/tlc-mass-spectrometry.html

Menadione-HydrazoneIn

tens

ity,

cps

m/z, Da

O

N

NH

NO2

NO2

Expected mass: 352.0808Observed mass: 351.075

Mass spectrometry:Collision-induced dissociation

(CID)

Parent ion Inert Gas Secondary ions

M2-

M1-

M3-

Neutral MoleculesM-

CID: Menadione-Hydrazone

Inte

nsit

y, c

ps

m/z, Da

351.0745

109.0173

167.0107

O

N

NH

NO2

NO2

NO2

NO2

diminished parent ion351.0745

secondary ion

secondary ion

Pyruvate-HydrazoneIn

tens

ity,

cps

m/z, Da

N

HO

O

NH

NO2

NO2

267.0376

Expected mass: 268.0444Observed mass: 267.0376

CID: Pyruvate-HydrazoneIn

tens

ity,

cps

m/z, Da

182.0213NH2

NO2

NO2

122.0252 NO2diminished parent ion

secondary ion

secondary ion

Mass Spectrometry Summary

Hydrazone Parent ion (m/z)

Secondary ions (m/z)

menadione 351.0745 167.0107, 109.0173

pyruvate 267.0376 182.0213, 122.0252

α-ketoglutarate 325.0436 182.0211, 152.0227, 122.0247

4-hydroxynonenal

335.1372 182.0218, 167.0110, 122.0252

The strongest candidates for a signature fragment from the DNPH-hydrazones are 182 and 122 m/z

NH2

NO2

NO2

Exact Mass: 182.0207

NO2

Exact Mass: 122.0248

NNH

NO2

NO2

R

Generic DNPH-hydrazone

Secondary Ion182 m/z

Secondary Ion122 m/z

Proposed structures for secondary ions

ConclusionPromising 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

AcknowledgementsDr. Gary MerrillDr. Kevin AhernDr. Fred StevensDr. Jong-Tae Yang

Jeff MorreAmira Barkal

OSU Mass Spectrometry LaboratoryHoward Hughes Medical InstituteCripps Undergraduate Research

Oregon State University