We generated a comprehensive picture of protease substrates in anti-Fas–treated apoptotic human Jurkat T lymphocytes. We used combined fractional diagonal chromatography (COFRADIC) sorting of protein amino-terminal peptides coupled to oxygen-16 or oxygen-18 differential labeling. We identified protease substrates and located the exact cleavage sites within processed proteins. Our analysis yielded 1,834 protein identifications and located 93 cleavage sites in 71 proteins. Indirect evidence of apoptosis-specific cleavage within 21 additional proteins increased the total number of processed proteins to 92. Most cleavages were at caspase consensus sites; however, other cleavage specificities suggest activation of other proteases. We validated several new processing events by immunodetection and by an in vitro assay using recombinant caspases and synthetic peptides containing presumed cleavage sites. The spliceosome complex appeared a preferred target, as 14 of its members were processed. Differential isotopic labeling further revealed specific release of nucleosomal components from apoptotic nuclei.
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The authors thank P. Vandenabeele for his critical reading of the manuscript, for the suggestions made and for providing the recombinant caspases. K.G. is a postdoctoral fellow and L.M. is a research assistant of the Fund for Scientific Research-Flanders (Belgium; F.W.O.-Vlaanderen). The project was supported by research grants from the Fund for Scientific Research-Flanders (Belgium; project number G.0008.03), the Inter University Attraction Poles (IUAP, project number P5/05), the GBOU research initiative (project number 20204) of the Flanders Institute of Science and Technology (IWT) and the European Union Interaction Proteome (6th Framework Program).
The authors declare no competing financial interests.
Histogram showing the distribution of ratios of isotopic peptide couples measured following COFRADIC isolation. (PDF 108 kb)
Analysis of the apoptotic-specific cleavage sites within known or predicted domains and motifs of the identified processed Jurkat proteins. (PDF 1648 kb)
N-termini of mitochondrial proteins. (PDF 89 kb)
Internally located α-N-acetylated peptides. (PDF 171 kb)
Identification features of peptides linked to proteins processed in apoptotic Jurkat cells. (PDF 149 kb)
Peptides only present in lysates of living cells. (PDF 128 kb)
Summary of proteins that are specifically cleaved in Fas-stimulated Jurkat T-lymphocytes. (PDF 113 kb)
Peptides exclusively found in the proteome digest of apoptotic cells. (PDF 102 kb)
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