Mass spectrometry-assisted protease substrate screening
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Mass spectrometry-assisted protease substrate screening. / Schlüter, Hartmut; Rykl, Jana; Thiemann, Joachim; Kurzawski, Sandra; Gobom, Johan; Tepel, Martin; Zidek, Walter; Linscheid, Michael.
In: ANAL CHEM, Vol. 79, No. 3, 01.02.2007, p. 1251-5.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Mass spectrometry-assisted protease substrate screening
AU - Schlüter, Hartmut
AU - Rykl, Jana
AU - Thiemann, Joachim
AU - Kurzawski, Sandra
AU - Gobom, Johan
AU - Tepel, Martin
AU - Zidek, Walter
AU - Linscheid, Michael
PY - 2007/2/1
Y1 - 2007/2/1
N2 - 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.
AB - 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.
KW - Blood Proteins
KW - Fibrinogen
KW - Humans
KW - Peptide Hydrolases
KW - Peptides
KW - Substrate Specificity
KW - Tandem Mass Spectrometry
KW - Thrombin
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
U2 - 10.1021/ac061482l
DO - 10.1021/ac061482l
M3 - SCORING: Journal article
C2 - 17263361
VL - 79
SP - 1251
EP - 1255
JO - ANAL CHEM
JF - ANAL CHEM
SN - 0003-2700
IS - 3
ER -