Identification of the endothelial cell microproteome. [ext224]

However, the study of smORF-encoded miPs has been hampered by technical challenges associated with the detection of small peptides by mass spectrometry. Here we combined deep RNA sequencing of ribosome-associated transcripts and optimized bioinformatic pipelines for downstream proteomic studies to identify thousands of smORF-derived miPs in endothelial cells (human 2,256, murine 7,597).

Profound alterations in smORF/miP expression were detected following human endothelial cell activation by interleukin 1beta (IL-1beta) in vitro and by hypercholesterolemia and altered blood flow in vivo (in mice). Genome-wide homology analyses revealed that human smORFs are highly conserved among primates, and poorly in rodents or zebrafish. Functional characterization of selected miPs demonstrated their physical interaction with proteins in different subcellular compartments and their involvement in the regulation of proliferation, cell death, gene expression and inflammation.