posted on 2020-04-03, 15:07authored byMari Enoksson, Jingwei Li, Melanie M. Ivancic, John C. Timmer, Eric Wildfang, Alexey Eroshkin, Guy S. Salvesen, W. Andy Tao
The identification of natural substrates and their cleavage sites is pivotal to defining proteolytic
pathways. Here we report a novel strategy for the identification of the signature of proteolytic cleavage
events based on quantitative proteomics. Lysine residues in proteins are blocked by guanidination so
that free N-terminals can be labeled with amine-specific iTRAQ reagents. The quantitative nature of
iTRAQ reagents allows us to distinguish N-terminals newly formed by proteolytic treatment (neoepitopes)
from original N-terminals in proteins. Proteins are digested with trypsin and analyzed using MALDI-TOF/TOF mass spectrometry. Peptides labeled with iTRAQ reagents are distinguished from other
peptides by exhibiting intense signature ions in tandem mass spectrometry analysis. A corresponding
data acquisition strategy was developed to specifically analyze iTRAQ tagged N-terminal peptides. To
validate the procedure, we examined a set of recombinant Escherichia coli proteins that have predicted
caspase-3 cleavage motifs. The protein mixture was treated with active or inactive caspase-3 and
subsequently labeled with two different iTRAQ reagents. Mass spectrometric analysis located 10
cleavage sites, all corresponding to caspase-3 consensus. Spiking caspase-cleaved substrate into a
human cell lysate demonstrated the high sensitivity of the procedure. Moreover, we were able to identify
proteolytic cleavage products associated with the induction of cell-free apoptosis. Together, these data
reveal a novel application for iTRAQ technology for the detection of proteolytic substrates.
Keywords: quantitative proteomics • protease • tandem mass spectrometry • caspases • MALDI • isotope labeling