期刊
JOURNAL OF PROTEOME RESEARCH
卷 6, 期 7, 页码 2850-2858出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr0701052
关键词
quantitative proteomics; protease; tandem mass spectrometry; caspases; MALDI; isotope labeling
资金
- NCRR NIH HHS [RR19752, RR20843] Funding Source: Medline
- NHLBI NIH HHS [N01-HV-28179] Funding Source: Medline
- DIVISION OF HEART AND VASCULAR DISEASES [N01HV028179] Funding Source: NIH RePORTER
- NATIONAL CENTER FOR RESEARCH RESOURCES [R21RR019752, U54RR020843] Funding Source: NIH RePORTER
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.
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