Quantitative proteome analysis of CD95 (Fas/Apo-1)-induced apoptosis by stable isotope labeling with amino acids in cell culture, 2-DE and MALDI-MS.
Proteome analysis of Jurkat T cells induced to undergo apoptosis by CD95 (Fas/Apo-1) treatment was performed to identify modified proteins. We used stable isotope labeling with amino acids in cell culture (SILAC) using leucine to identify proteins of apoptotic and control Jurkat T cells by 2-DE and MALDI-MS. Out of 224 spots analyzed, we quantified 213 spots with 3.5 leucine-containing peptide pairs on average; 28 proteins with a relative abundance of higher than 1.5 were found. Five new modified proteins including calcyclin binding protein, cytosolic acyl coenzyme A thioester hydrolase, heterogeneous ribonucleoprotein M, replication factor C 37-kDa subunit, and tropomyosin 4 chain were identified as being modified in response to apoptosis. In comparison to differential proteome analysis via silver-stained 2-D gels and PMF of total Jurkat T cell lysates, 15 additional apoptosis-modified proteins were identified though 8 proteins were not found. The described approach using SILAC instead of silver staining for relative quantification was simpler to perform regarding the number of required 2-D gels, that cumbersome gel comparisons were avoided, and more apoptosis-modified proteins were identified, but with a higher demand on data interpretation of the mass spectra obtained.
Thiede
Bernd
B
Max Planck Institute for Infection Biology, Department Molecular Biology, Berlin, Germany.
Kretschmer
Annikki
A
Rudel
Thomas
T
D017209:Apoptosis; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D019169:Jurkat Cells; D007930:Leucine / Q000737:chemistry / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019014:fas Receptor / Q000378:metabolism*
D020543:Proteome; D019014:fas Receptor; D007930:Leucine