Abstract

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments.

Highlights

  • The vasculature has an important role in embryonic development and tissue homeostasis [1]

  • The quantification values obtained (PAI) for all proteins in chorioallantoic membrane (CAM), intestine, kidney, and liver samples were transformed into a matrix that was used in the R software to organize the proteins into cluster groups

  • Novel Approach—In this research, we applied in vivo biotinylation combined with high-resolution mass spectrometry and bioinformatic analyses to study the vascular and matrix proteome in the chicken embryo

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Summary

EXPERIMENTAL PROCEDURES

Tissue Biotinylation—Brown Leghorn eggs were cultured at 38 °C for 3 days; shells were cracked and the egg contents were transferred to 10-cm-diameter cell culture dishes. The software uses the validated identification results and extracted ion chromatograms (XICs) of the identified peptide ions in the corresponding raw nano-LC-MS files, based on their experimentally measured retention time and monoisotopic m/z values. If some peptide ions were sequenced via MS/MS and validated only in some of the samples to be compared, their XIC signal was extracted in the nanoLC-MS raw file of the other samples using a predicted retention time value calculated from this alignment matrix via a linear interpolation method. The quantification values obtained (PAI) for all proteins in CAM, intestine, kidney, and liver samples were transformed into a matrix that was used in the R software to organize the proteins into cluster groups. A detailed description of the materials and methods can be found in the supplemental “Experimental Procedures” section

RESULTS
CC annotation
Stress proteins
EndoFactor positive
DISCUSSION
Human peptides
Gene Ontology cell component
Full Text
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