"Reverse degradomics", monitoring of proteolytic trimming by multi-CE and confocal detection of fluorescent substrates and reaction products.
A platform for profiling of multiple proteolytic activities acting on one specific substrate, based on the use of a 96-channel capillary DNA sequencer with CE-LIF of labeled substrate peptides and reaction products is introduced. The approach consists of synthesis of a substrate peptide of interest, fluorescent labeling of the substrate, either aminoterminally by chemical coupling, or carboxyterminally by transglutaminase reaction, proteolysis by a biological mixture of proteases in the absence or presence of protease inhibitors, multi-channel analysis of substrate and reaction products, and data collection and processing. Intact substrate and reaction products, even when varying by only one amino acid, can be relatively semi-quantified in a high-throughput manner, yielding information on proteases acting in complex biological mixtures and without prepurification. Monitoring, classification and inhibition of multiple proteolytic activities are demonstrated on a model substrate, the aminoterminus of the mouse granulocyte chemotactic protein-2. In view of extensive processing of chemokines into various natural forms with different specific biological activities, and of the fragmentary knowledge of processing proteases, examples of processing by neutrophil degranulate, tumor cell culture fluids and plasma are provided. An example of selection and comparison of inhibitory mAbs illustrates that the platform is suitable for inhibitor screening. Whereas classical degradomics technologies analyze the substrate repertoire of one specific protease, here the complementary concept, namely the study of all proteases acting, in a biological context, on one specific substrate, is developed and tuned to identify key proteases and protease inhibitors for the processing of any biological substrate of interest.
Piccard
Helene
H
Laboratory of Immunobiology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.
Hu
Jialiang
J
Fiten
Pierre
P
Proost
Paul
P
Martens
Erik
E
Van den Steen
Philippe E
PE
Van Damme
Jo
J
Opdenakker
Ghislain
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D054427:Chemokine CXCL6 / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods*; D010447:Peptide Hydrolases / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000032:analysis*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D011503:Transglutaminases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D054427:Chemokine CXCL6; D005456:Fluorescent Dyes; D061965:Matrix Metalloproteinase Inhibitors; D019151:Peptide Library; D010455:Peptides; D011503:Transglutaminases; D010447:Peptide Hydrolases