Identification of Carboxypeptidase Substrates by C-Terminal COFRADIC.
We here present a detailed procedure for studying protein C-termini and their posttranslational modifications by C-terminal COFRADIC. In fact, this procedure can enrich for both C-terminal and N-terminal peptides through a combination of a strong cation exchange fractionation step at low pH, which removes the majority of nonterminal peptides in whole-proteome digests, while the actual COFRADIC step segregates C-terminal peptides from N-terminal peptides. When used in a differential mode, C-terminal COFRADIC allows for the identification of neo-C-termini generated by the action of proteases, which in turn leads to the identification of protease substrates. More specifically, this technology can be applied to determine the natural substrate repertoire of carboxypeptidases on a proteome-wide scale.
VIB-UGent Center for Medical Biotechnology, B-9000, Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000, Ghent, Belgium.
Sebastian
S
Tanco
Institut de Biotecnologia i Biomedicina (IBB), Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, IBB-Campus de la UAB, Bellaterra, 08193, Barcelona, Spain.
Francesc Xavier
FX
Aviles
VIB-UGent Center for Medical Biotechnology, B-9000, Ghent, Belgium.|Department of Biochemistry, Ghent University, B-9000, Ghent, Belgium.
Kris
K
Gevaert
Institut de Biotecnologia i Biomedicina (IBB), Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, IBB-Campus de la UAB, Bellaterra, 08193, Barcelona, Spain. julia.lorenzo@uab.es.
Julia
J
Lorenzo
VIB-UGent Center for Medical Biotechnology, B-9000, Ghent, Belgium. petra.vandamme@vib-ugent.be.|Department of Biochemistry, Ghent University, B-9000, Ghent, Belgium. petra.vandamme@vib-ugent.be.
Petra
P
Van Damme
D002268:Carboxypeptidases / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
D010455:Peptides; D020543:Proteome; D002268:Carboxypeptidases
C-terminomics; COFRADIC; Carboxypeptidases; Degradomics; Proteases; Protein C-termini; Substrates