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Shotgun proteome analysis of protein cleavage in apoptotic cells
Author(s) -
Thiede Bernd,
Treumann Achim,
Kretschmer Annikki,
Söhlke Jana,
Rudel Thomas
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401110
Subject(s) - shotgun proteomics , proteome , shotgun , gel electrophoresis , apoptosis , proteomics , nucleolin , jurkat cells , trypsin , chemistry , microbiology and biotechnology , polyacrylamide gel electrophoresis , tandem mass spectrometry , protease , biochemistry , biology , chromatography , mass spectrometry , enzyme , genetics , gene , immune system , t cell , cytoplasm , nucleolus
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.