Towards a standardized human proteome database: Quantitative proteome profiling of living cells

• Published in: Proteomics (Weinheim) Vol. 4; no. 5; pp. 1314 - 1323
• Main Authors: Traxler, Elisabeth, Bayer, Editha, Stöckl, Johannes, Mohr, Thomas, Lenz, Christof, Gerner, Christopher
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.05.2004; WILEY‐VCH Verlag
• Subjects: Autoradiography; Cells, Cultured; Databases, Factual - standards; Electrophoresis, Gel, Two-Dimensional; Endothelial cells; Endothelium, Vascular - chemistry; Endothelium, Vascular - drug effects; Fluorescence; Fluorescence detection; Humans; Jurkat Cells; Kinetics; Leukaemia; Lymphocyte Activation; Peptide Fragments - analysis; Phytohemagglutinins - pharmacology; Proteins - chemistry; Proteins - metabolism; Proteome - analysis; Proteome - chemistry; Proteome - metabolism; Sensitivity and Specificity; Subcellular Fractions; Sulfur Radioisotopes - metabolism; T-Lymphocytes - chemistry; T-Lymphocytes - drug effects; Trypsin; Two-dimensional gel electrophoresis; Umbilical Veins - cytology; Vascular Endothelial Growth Factor A - drug effects
• DOI: 10.1002/pmic.200300774
• ISSN: 1615-9853

Comparative proteome profiling, performed by two‐dimensional polyacrylamide gel electrophoresis or multidimensional protein identification technology, usually relies on the relative comparison of samples of interest with respect to a reference. Currently, no standardized quantitative protein expression database of human cells, facilitating data comparisons between different laboratories, exists. Recently, we have published two‐dimensional polyacrylamide gel electrophoresis‐based techniques to assess absolute protein data comprising protein amounts, synthesis rates and biological half‐lives (Mol. Cell. Proteomics 2002, 1, 528–537). Determination of protein amounts by fluorography of two‐dimensional gels was followed by the exact quantification of the amount of incorporated 35S radiolabel. Here we demonstrate an application of this highly standardized method to quiescent human T cells, phythaemagglutinin‐stimulated T cells and Jurkat cells, a human T lymphoblast cell line. While the protein composition of quiescent T cells differed significantly compared to that of Jurkat cells, it was only slightly different compared to the activated T cells. Synthesis profile analyses demonstrated that activated T cells clearly differed from the quiescent cells, performing apparently almost like lymphoblast cells. The great sensitivity of this approach was further demonstrated with human umbilical vein endothelial cells treated for six hours with vascular endothelial growth factor. While no significant alteration of protein amounts was detected at all upon activation, the synthesis rate of several proteins was found to be more than doubled.