Mass spectrometry-assisted protease substrate screening.
Since sequencing of the human genome was completed, more than 500 genes have been annotated as proteases. Exploring the physiological role of each protease requires the identification of their natural substrates. However, the endogenous substrates of many of the human proteases are as yet unknown. Here we describe a new assay that addresses this problem. The assay, which easily can be automated, is based on the incubation of immobilized protein fractions, which may contain the natural substrate, with a defined protease. After concentrating the proteolytically released peptides by reversed-phase chromatography they are analyzed by tandem mass spectrometry and the substrates identified by database searching. The proof of principle in this study is demonstrated by incubating immobilized human plasma proteins with thrombin and by identifying by tandem mass spectrometry the fibrinopeptides, released by the action of thrombin from their natural substrate fibrinogen, in the reaction mixture.
Schlüter
Hartmut
H
Med. Clinic IV, Nephrology, Campus Benjamin Franklin, Charité-University Medicine Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. Hartmut.schlueter@charite.de
Rykl
Jana
J
Thiemann
Joachim
J
Kurzawski
Sandra
S
Gobom
Johan
J
Tepel
Martin
M
Zidek
Walter
W
Linscheid
Michael
M
D001798:Blood Proteins; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D010447:Peptide Hydrolases / Q000097:blood* / Q000378:metabolism; D010455:Peptides / Q000032:analysis*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013917:Thrombin / Q000378:metabolism
D001798:Blood Proteins; D010455:Peptides; D005340:Fibrinogen; D010447:Peptide Hydrolases; D013917:Thrombin