Rapid profiling of peptide stability in proteolytic environments.
The notorious degradation susceptibility of peptides is a major obstacle to their use as medicinal drugs. Assays with which the stability of peptides in complex proteolytic environments can be determined are thus indispensable for peptide drug development. Herein, we describe a new peptide proteolysis assay that meets that demand. It unites the high-throughput capacity of heterogeneous with the well-defined kinetic characteristics of homogeneous assay formats and operates on the cleavage-caused loss of a detection handle. We have confirmed the assay's accuracy with well-defined model interactions and proved its high versatility and robustness with a representative application where we determined the half-lives of 375 different peptides in a crude intestinal protease preparation. With this reliable, reproducible, and efficient assay the enzyme kinetics of any peptide-protease interaction is accessible even for complex protease solutions.
Gorris
Hans H
HH
Division of Mucosal Immunology, Research Center Borstel, Parkallee 22, 23845 Borstel, Germany.
Bade
Steffen
S
Röckendorf
Niels
N
Albers
Eike
E
Schmidt
M Alexander
MA
Fránek
Milan
M
Frey
Andreas
A
D000595:Amino Acid Sequence; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D006207:Half-Life; D007421:Intestine, Small / Q000201:enzymology; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D011137:Polystyrenes / Q000737:chemistry; D055550:Protein Stability; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D019151:Peptide Library; D010455:Peptides; D011137:Polystyrenes; D010447:Peptide Hydrolases