title: Identification of proteolytic cleavage sites by quantitative proteomics.
abstract: 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.
authors.affiliation: Burnham Institute for Medical Research, La Jolla, California 92037, USA.
authors.forename: Mari
authors.initials: M
authors.lastname: Enoksson
authors.forename: Jingwei
authors.initials: J
authors.lastname: Li
authors.forename: Melanie M
authors.initials: MM
authors.lastname: Ivancic
authors.forename: John C
authors.initials: JC
authors.lastname: Timmer
authors.forename: Eric
authors.initials: E
authors.lastname: Wildfang
authors.forename: Alexey
authors.initials: A
authors.lastname: Eroshkin
authors.forename: Guy S
authors.initials: GS
authors.lastname: Salvesen
authors.forename: W Andy
authors.initials: WA
authors.lastname: Tao
mesh_terms: D000595:Amino Acid Sequence; D053148:Caspase 3 / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; D053148:Caspase 3; D008239:Lysine