Protein processing characterized by a gel-free proteomics approach.
We describe a method for the specific isolation of representative N-terminal peptides of proteins and their proteolytic fragments. Their isolation is based on a gel-free, peptide-centric proteomics approach using the principle of diagonal chromatography. We will indicate that the introduction of an altered chemical property to internal peptides holding a free alpha-N-terminus results in altered column retention of these peptides, thereby enabling the isolation and further characterization by mass spectrometry of N-terminal peptides. Besides pointing to changes in protein expression levels when performing such proteome surveys in a differential modus, protease specificity and substrate repertoires can be allocated since both are specified by neo-N-termini generated after a protease cleavage event. As such, our gel-free proteomics technology is widely applicable and amenable for a variety of proteome-driven protease degradomics research.
Van Damme
Petra
P
Ghent University, Ghent, Belgium.
Impens
Francis
F
Vandekerckhove
Joël
J
Gevaert
Kris
K
D000818:Animals; D002460:Cell Line; D030562:Databases, Protein; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010103:Oxygen Isotopes / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*
D010103:Oxygen Isotopes; D010446:Peptide Fragments