Quantitative PCR-based approach for rapid phage display analysis: a foundation for high throughput vascular proteomic profiling.
Functional proteomic strategies offer unique advantages over current molecular array approaches, as the epitopes identified can directly provide bioactive peptides for investigational and/or translational applications. The vascular endothelium is well suited to proteomic assessment by in vivo phage display, but extensive enrichment and sequencing steps limit its application for high throughput molecular profiling. To overcome these limitations we developed a quantitative PCR (Q-PCR) strategy to allow the rapid quantification of in vivo phage binding. Primers were designed for distinct clones selected from a defined phage pool to probe for age-associated changes in cardiac vascular epitopes. Sensitivity and specificity of the primer sets were tested and confirmed in vitro. Q-PCR quantification of phage in vivo confirmed the preferential homing of all phage clones to the young rather than old cardiac vasculature and demonstrated a close correlation with phage measurements previously determined using traditional bacterial-based titration methods. This Q-PCR approach provides quantification of phage within hours of phage injection and may therefore be used for rapid, high throughput analysis of binding of defined phage sequences both in vivo and in vitro, complementing nonbiased phage approaches for the proteomic mapping of vascular beds and other tissues.
Ballard
Victoria L T
VL
Department of Medicine and Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York 10021, USA.
Holm
Jacquelyne M
JM
Edelberg
Jay M
JM
D000375:Aging; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D003331:Coronary Vessels / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000235:genetics; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D010446:Peptide Fragments; D019151:Peptide Library