Identification of Proteolytic Cleavage Sites by Quantitative Proteomics

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