Analysis of carbonyl-containing compounds derivatized

Report
Analysis of carbonylcontaining 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
Carbonyls are a highly
reactive species
Build up of carbonyls
cause carbonyl stress
Pyruvate
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
Dinitrophenylhydrazine (DNPH)
menadione-hydrazone
menadione
DNPH
DNPH is a good derivatizing agent for stabilizing
carbonyls
Primary amine attaches to the carbonyl and forms
a hydrazone
Hydrazones
Some hydrazones form precipitates
Precipitates dissolve in different solvents
depending on the hydrazone
Hydrazone
Formed in
Precipitates
Menadione
1 M HCl,
Yes
23.8% EtOH
Chloroform Blue
Pyruvate
0.5 M HCl
Yes
95% EtOH
Brown
α-Ketoglutarate
0.5 M HCl, Yes
47.5% EtOH
95% EtOH
Brown
n/a
Brown
4-Hydroxynonenal 0.5 M HCl, No
47.5% EtOH
Dissolves in Color under
basic conditions
Thin layer chromatography
Fig. 2. TLC analysis of
DNPH, menadione,
and menadionehydrazone. 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
menadionehydrazone
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-Hydrazone
Intensity, cps
Expected mass: 352.0808
Observed mass: 351.075
m/z, Da
Mass spectrometry:
Collision-induced dissociation (CID)
M2M1M3MParent ion
Neutral Molecules
Inert Gas
Secondary ions
CID: Menadione-Hydrazone
Intensity, cps
109.0173
secondary ion
167.0107
secondary ion
m/z, Da
diminished
351.0745
parent ion
351.0745
Pyruvate-Hydrazone
Intensity, cps
267.0376
Expected mass: 268.0444
Observed mass: 267.0376
m/z, Da
CID: Pyruvate-Hydrazone
Intensity, cps
182.0213 secondary ion
secondary
ion
122.0252
diminished
parent ion
m/z, Da
Mass Spectrometry Summary
The strongest candidates for a signature fragment from
the DNPH-hydrazones are 182 and 122 m/z
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
Proposed structures for
secondary ions
Generic
DNPH-hydrazone
Secondary Ion
182 m/z
Secondary Ion
122 m/z
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

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