Shotgun proteome analysis of protein cleavage in apoptotic cells.
A new shotgun proteomics approach was employed to identify degraded proteins. Jurkat T-cells were induced to undergo apoptosis by Fas (CD95/Apo-1) stimulation. The proteins were separated by large (30 cm) sodium dodecyl sulphate-polyacrylamide gel electrophoresis and identified by liquid chromatography-tandem mass spectrometry after digestion of 100 gel slices with trypsin. The molecular masses of the individual gel slices were calculated through the known theoretical masses of the identified proteins. Proteins were defined as degradation candidates if either the empirical determined molecular mass was at most 80% of the theoretical value, or if proteins were identified in clearly different gel slices. In this manner, the degradation of 11 already identified apoptosis-modified proteins was confirmed and nine until now unknown degradation candidate proteins identified. Degradation during apoptosis must be verified by additional techniques such as in vitro caspase assays as shown for nucleolin and Rho GDI 2. The results presented confirm the suitability of a shotgun approach for the identification of putative protease targets.
Thiede
Bernd
B
Department Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
Treumann
Achim
A
Kretschmer
Annikki
A
Söhlke
Jana
J
Rudel
Thomas
T
D017209:Apoptosis; D020169:Caspases / Q000032:analysis / Q000378:metabolism; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D013058:Mass Spectrometry; D010750:Phosphoproteins / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000502:physiology*; D040901:Proteomics / Q000379:methods*; D016601:RNA-Binding Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000473:pathology / Q000502:physiology*; D025521:Tumor Suppressor Proteins; D019014:fas Receptor / Q000502:physiology; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D000097796:Nucleolin
D020169:Caspases; D020730:Guanine Nucleotide Dissociation Inhibitors; D010750:Phosphoproteins; D011506:Proteins; D016601:RNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D019014:fas Receptor; D062949:rho Guanine Nucleotide Dissociation Inhibitor beta; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D000097796:Nucleolin; C569658:ARHGDIB protein, human