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

Abstract

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.

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Figure 1: Quality analysis of rat lung P and RLMVEC P by western blot analysis.
Figure 2: Reproducibility of protein identification in rat lung P.
Figure 3: Spectrum coverage per protein and physicochemical properties.
Figure 4: Classification of proteins identified in rat lung P.
Figure 5: Analysis of identified proteins.
Figure 6: Protein validation.

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Acknowledgements

We thank Michelle Bourne, Lisa Randall and Traci Smith for technical assistance; David Tabb for help setting up DTASelect; and Yan Li for helpful discussion. This research was supported by grants to J.E.S from the National Institutes of Health (Heart, Lung and Blood nos. R01 HL52766, R01 HL58216), National Cancer Institute (no. R01 CA83989, R24 CA095893, R33 CA97528), Sidney Kimmel, Schutz Foundation, California Tobacco-related Disease Research Program (no. 11RT-0167) and California Breast Cancer Research Program (no. 8WB-00114).

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Correspondence to Jan E Schnitzer.

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Supplementary information

Supplementary Table 1

Integral and lipid-anchored plasma membrane proteins (PDF 203 kb)

Supplementary Table 2

Endothelial cell associated marker proteins (PDF 173 kb)

Supplementary Notes

Additional findings and validation (PDF 338 kb)

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Durr, E., Yu, J., Krasinska, K. et al. Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture. Nat Biotechnol 22, 985–992 (2004). https://doi.org/10.1038/nbt993

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