Macfarlane Research Group
Ronald Macfarlane PhD., University Distinguished Professor, D’Vesharronne Moore, Craig Larner, Paul Cammarata
Laboratory for Cardiovascular Chemistry, Department of Chemistry, Texas A&M University
Lipoprotein Fingerprinting: A link between Analytical Chemistry
And Healthcare for the Diagnosis of Coronary Heart Disease
OBJECTIVE: To Develop a Combination of Chemical Methods for the Diagnosis of Early Onset of Cardiovascular Disease
Difference in Apolipoprotein C1 Found for CVD Samples Vs Controls:
High Performance Lipoprotein Profiling:
Forming the Lipoprotein Fingerprint:
Using a Drop of
Blood
Equilibrium Density Gradient
Ultracentrifugation
H4EDTA
+ CdCO3
MALDI Mass Spectrometry
Control HDL-2
CVD HDL-2
Control HDL-3
CVD HDL-3
Density Gradient Theory
→ H2CdEDTA + H2O + CO2
H2CdEDTA + Cs2CO3 → Cs2CdEDTA + H2O + CO2
+
Mass shift found for Apo C1 for
all CVD patients in a small
sample study. For these
samples, the apoptosis values
were also found to be 50%
higher. In contrast, the Control
patients had normal Apo C1
with lower apoptosis values.
This indicates a possible
connection between the Apo C1
and the development of CHD.
Further Testing of Lipoprotein Fingerprint Samples:
Chemical and Immunoaffinity
Cell Culture Studies
Integrated Intensities from the Lipoprotein Fingerprint
Lipoprotein Finger Print
Sample
TRL
LDL-1 LDL-2 LDL-3 LDL-4
LDL5 HDL-2b HDL-2a HDL-3a HDL-3b HDL-3c
Serum added to
Substance A and
shaken to form
lipoprotein bound
complex and spun
to obtain
supernatant
Isoelectric Focusing
-
+
-
Supernatant
consists of
serum depleted
of lipoprotein or
protein of
interest
Stripping buffer
is added to
beads to elute
Lipoprotein or
protein of interest
and spun to obtain
supernatant
Supernatant
consists of
isolated
lipoprotein or
protein
of interest
Capillary Electrophoresis
+
Delipidated
Denatured Proteins
IPG Gel
Children’s Study
CVD Separation
7770 & 1532C CVD Library
Study (90 Patients)
-
pH=7
+
Enhanced Separation Of
Normal Lipodemic Profiles
Statistical Separations
pH=4
Focused Proteins
pH=4
pH=7
7770 and 2.5
1532C Control CVD Study
CVD (1) vs Control (2)
2
1.5
CVD
1
Control
0.5
0
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
1.5
LDA Separation Value
P-value = 0.14352
% Correct = 100%
P-value = 0.23306
% Correct = 100%
P-value = 0.03804
% Correct = 85.6%
2
2.5
Recent and Future Publications:
1. P.V. Bondarenko, Z.N. Farwig, C.J. McNeal, R.D. Macfarlane, MALDI- and ESI-MS of the HDL apolipoproteins; new isoforms of apoA-I, A-II, Int. J.
Mass Spectrom. Ion Process 219 (2002) 671–680
2. J.D. Johnson, N.J. Bell, E.L. Donahue, R.D. Macfarlane, Metal ion complexes of EDTA as solutes for density gradient ultracentrifugation: influence of
metal ions, Anal. Chem. 77 (2005) 7054–7061
3. Henriquez, R.R. et al. World Intellectual Property Organization (2009) Characterization of Biological Samples. Patent # WO/2009/152437
4. D. Moore et al. Isoforms of apolipoprotein C-I with Individuals with Coronary Artery Disease. Biochemical and Biophysical Research Communications
404 (2011) 1034–1038
5. Larner, C., Macfarlane, R.D. High Performance Lipoprotein Profiling. Anal. Chem. (To Be Published)
6. Larner, C., Cammarata, P., Ortega, E., Macfarlane, R.D. Use of Lipoprotein Fingerprints and Statistics to Classify Any Subjects for Coronary Artery
Disease . Anal. Chem. (To Be Published)
7. Cammarata, P., Larner, C., Macfarlane, R.D. Lipoprotein Fingerprint: A New Approach to Classifying Normal Lipidemic Subjects for Coronary Artery
Disease. Anal. Chem. (To Be Published)
8. Cammarata, P., Macfarlane, R.D. An Accurate and Precise Fraction Collector for Preparative Lipoprotein Density Profiling. Anal. Chem. (To Be
Published)