Assays of endogenous caspase activities: a comparison of mass spectrometry and fluorescence formats.
This paper describes a label-free assay for measuring endogenous caspase protease activities in cell lysates. The assay format, termed SAMDI-MS (self-assembled monolayers for matrix assisted laser desorption ionization time-of-flight mass spectrometry), is based on the enzymatic modification of peptides immobilized to monolayer substrates, followed by direct detection of the products with mass spectrometry. Monolayers presenting peptide substrates for either caspase-3 or -8 were treated with lysates from Jurkat cells that were stimulated with staurosporine and SKW6.4 cells that were stimulated with LzCD95L. In both cases, the SAMDI assays reported on the activation of endogenous caspase enzymes with levels of detection that are similar to those observed using the commonly employed fluorogenic assays. The use of longer peptide substrates, which are not compatible with the fluorogenic assays, provided for a better resolution of the two caspase activities. This work is significant because it demonstrates that the SAMDI assay can be used to measure endogenous enzyme activities and because it avoids the loss of activity and specificity that often accompany label-dependent assay formats.
Su
Jing
J
Howard Hughes Medical Institute and Department of Chemistry, University of Chicago, Chicago, Illinois 60637, USA.
Rajapaksha
Tharinda W
TW
Peter
Marcus E
ME
Mrksich
Milan
M
D000595:Amino Acid Sequence; D017209:Apoptosis; D053148:Caspase 3 / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D053181:Caspase 8 / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation* / Q000379:methods*; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
D053148:Caspase 3; D053181:Caspase 8