Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture

Endothelial cells can function differently in vitro and in vivo; however, the degree of microenvironmental modulation in vivo remains unknown at the molecular level largely because of analytical limitations. We use multidimensional protein identification technology (MudPIT) to identify 450 proteins (with three or more spectra) in luminal endothelial cell plasma membranes isolated from rat lungs and from cultured rat lung microvascular endothelial cells. Forty-one percent of proteins expressed in vivo are not detected in vitro. Statistical analysis measuring reproducibility reveals that seven to ten MudPIT measurements are necessary to achieve ≥95% confidence of analytical completeness with current ion trap equipment. Large-scale mapping of the proteome of vascular endothelial cell surface in vivo, as demonstrated here, is advisable because distinct protein expression is apparently regulated by the tissue microenvironment that cannot yet be duplicated in standard cell culture.