Article

Development of inductively coupled plasma-mass spectrometry-based protease assays.

Department of Chemistry, University of Toronto, Toronto, Ont., Canada M5S 3H6.
Analytical Biochemistry (impact factor: 3). 11/2009; 398(1):93-8. DOI:10.1016/j.ab.2009.11.010 pp.93-8
Source: PubMed

ABSTRACT Rapid, sensitive, and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here an assay for protease activity that uses inductively coupled plasma-mass spectrometry (ICP-MS) detection is described. Peptidic alpha-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N terminus to provide a distinct elemental tag. A biotin label was appended to the C terminus of the peptide, allowing separation of uncleaved peptide from the enzymatic digestion. The enzyme activity was determined by quantifying the lanthanide ion signal of the peptide cleavage products by ICP-MS. Biotinylated substrates synthesized include Lu-DTPA-Asp-Leu-Leu-Val-Tyr approximately Asp-Lys(biotin) and Lu-DTPA-betaAla-betaAla-betaAla-betaAla-Gly-Ser-Ala-Tyr approximately Gly-Lys-Arg-Lys(biotin)-amide. Parallel assays with a commercially available fluorogenic substrate (Suc-AAPF-AMC) for alpha-chymotrypsin were performed for comparison. Using the ICP-MS assay, enzyme concentrations as low as 2pM could be readily detected, superior to the detection limit of an assay using the alpha-chymotrypsin fluorogenic substrate (Suc-AAPF-AMC). Furthermore, we demonstrated the use of this approach to detect chymotrypsin activity in HeLa cell lysates.

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Keywords

alpha-chymotrypsin fluorogenic substrate
 
biotin label
 
Biotinylated substrates synthesized
 
C terminus
 
commercially available fluorogenic substrate
 
detection limit
 
distinct elemental tag
 
drug development
 
enzyme concentrations
 
HeLa cell lysates
 
ICP-MS assay
 
lanthanide ion chelate
 
lanthanide ion signal
 
N terminus
 
Parallel assays
 
peptide cleavage products
 
Peptidic alpha-chymotrypsin substrates
 
quantitative assays
 
Rapid
 
uses inductively
 

Urja S Lathia