Methods for Mapping the Extracellular and Membrane Proteome in the Avian Embryo, and Identification of Putative Vascular Targets or Endothelial Genes.
Methods in molecular biology, 2018
Kilarski WW, Herbert J, BikfalviA.
We present a protocol for the specific labeling and isolation of proteins from the membrane surface of endothelial cells and the surrounding extracellular matrix of organs, experimental wounds and tumors using chicken embryos. Proteins are deglycosylated on streptavidin resin and purified after gentle elution and trypsin digestion. Peptides are analyzed by spectroscopy and reverse proteomic fingerprinting. The major advantages of this protocol include reductions in both the background and overrepresentation of single proteins that would otherwise mask less well-represented proteins in the mass spectroscopy analysis. We also present methods to identify putative vascular and endothelial cell targets from isolated chicken membranes and extracellular proteins. The use of human genome and transcriptome data facilitates this analysis. Human orthologs of isolated chicken proteins are identified using best hit BLAST searches against the Human Reference Sequence Database. The expression of Human orthologs is then assessed for endothelial and non-endothelial cell enrichment using second generation RNA-seq sequenced libraries. Scanning of the published literature then provides a ranking score of those genes most likely involved in cancer or having a link to angiogenesis.
Chorio-allantoic membrane; In vivo biotinylation; Mass spectrometry; Membrane and matrix proteome; Protein mapping; Proteomics chick embryo