Cardiovascular Proteomics
Tools to Develop Novel Biomarkers and Potential Applications
Sara Arab, PhD*,¶,
Anthony O. Gramolini, PhD*, ,
Peipei Ping, PhD#,
Thomas Kislinger, PhD , ,
Brian Stanley, PhD||,**,
Jennifer van Eyk, PhD||,**,
Maral Ouzounian, MD*,||,
David H. MacLennan, PhD*,,
Andrew Emili, PhD*, and
Peter P. Liu, MD*,¶,*
* Heart and Stroke/Richard Lewar Center of Excellence, University of Toronto and the Canadian Institutes of Health Research, Ottawa, Ontario, Canada
Department of Physiology
Banting and Best Department of Medical Research
Program in Proteomics and Bioinformatics, University of Toronto, Toronto, Ontario, Canada
|| Queen's University, Kingston, Ontario, Canada
¶ Department of Medicine, University of Toronto, Toronto, Ontario, Canada
# University of California Los Angeles–David Geffen School of Medicine, Los Angeles, California
** Johns Hopkins NHLBI Proteomics Center, Baltimore, Maryland
View larger version (63K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 1 Large-scale protein analysis of myocytes can be done with 2-dimensional gel electrophoresis (2-DE), where the proteins can be separated and identified on the basis of molecular weight and pH. However, to improve the resolution and to achieve greater protein separation, optimization of the gel processing conditions (e.g., expanding the pH range by selective isoelectric [IE] focusing) helps to improve the resolution significantly (for details, see Stanley et al. [37]). ASB = amidosulfobetaine; SDS PAGE = sodium dodecylsulfate polyacrylamide gel electrophoresis.
|
|
View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 2 "Shotgun" proteomic analysis combining liquid chromatography with mass spectrometry (LC-MS) or so-called multidimensional protein identification technology (MudPIT). In this approach, proteins are enzymatically digested and subjected to fractionation through cation exchange and reverse phase, and the peptide fragments are sprayed through high voltage into the mass spectrometer. The unique emerging spectra are then recorded and compared with existing databases to search for their identity. HPLC = high-pressure liquid chromatography; MS = mass spectometry; RP = reverse phase; SCX = strong cation exchange.
|
|
View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 3 Cluster analysis of mouse myocardial peptide fractions emerging from "shotgun" processing. The peptides naturally cluster into cytosolic, microsomal, mitochondrial, and nuclear fractions. The peptide localization confers functional specificity, such that the calcium regulatory proteins are much more abundant in the microsomal fraction, whereas transcription factors are in the nuclear-specific fractions. Ca2+ = calcium; GO = Gene Ontology; MudPIT = multidimensional protein identification technology.
|
|
View larger version (68K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 4 Comparison of microarray and proteomic informatic analysis of muscle cell development in culture. Red color denotes gene up-regulation, whereas green color indicates down-regulation. The result shows that there is a reasonable correspondence of genes seen in microarray and proteomics. However, there are also very unique species that are differentially regulated between gene expression and protein presence. This shows that the transcriptosomic and proteomic approaches are complementary and not duplicative. DNA = deoxyribonucleic acid; GO = Gene Ontology; mRNA = messenger ribonucleic acid. Reprinted with permission from Kislinger T, Gramolini A, Pan Y, et al. Proteome dynamics during C2Cl2 myoblast differentiation. Mol Cell Proteomics 2005;4:887–901.
|
|
View larger version (48K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 5 Screening or validation of protein peptide markers can again be conducted after sample separation with high-pressure liquid chromatography (HPLC), followed by mass spectrometer analysis. However, ability to remove overabundant species such as albumin or immunoglobulins will significantly facilitate the detection of smaller peptide species with higher sensitivity.
|
|
|
