Quantitative PCR-based approach for rapid phage display analysis: a foundation for high throughput vascular proteomic profiling.

Abstract:

: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.

journal_name

Physiol Genomics

journal_title

Physiological genomics

authors

Ballard VL,Holm JM,Edelberg JM

doi

10.1152/physiolgenomics.00025.2006

subject

Has Abstract

pub_date

2006-08-16 00:00:00

pages

202-8

issue

3

eissn

1094-8341

issn

1531-2267

pii

00025.2006

journal_volume

26

pub_type

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