Methods and systems for determining ADAMTS13 enzyme activity
Inventors
Grant, Russell Philip • Shuford, Christopher Michael • Bradley, Meghan Norris
Assignees
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Publication Number
US-12442030-B2
Publication Date
2025-10-14
Expiration Date
Abstract
Disclosed are methods and systems for the analysis activity of enzyme disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) in a sample. The methods and systems disclosed herein can be useful for diagnosis of thrombotic thrombocytopenic purpura in a patient.
Core Innovation
The described invention provides a system for determining activity of ADAMTS13 in a sample by using an exogenous peptide substrate that is cleavable by ADAMTS13. The system includes a station for incubating the sample with the exogenous peptide substrate under conditions allowing enzymatic cleavage to generate an enzymatic cleavage product, and the substrate consists of an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 3 and comprises an ADAMTS13 cleavage site.
After incubation, the system includes a station for ionizing the enzymatic cleavage product to generate a multiply charged gas-phase ion. The method then uses a station for analyzing the multiply charged gas-phase ion by tandem mass spectrometry to determine the presence and/or amount of the enzymatic cleavage product in the sample, where the presence or amount is indicative of the activity of ADAMTS13 in the sample.
In the described implementation, the cleavage product generated from a vWF-derived synthetic peptide substrate is detected by tandem mass spectrometry, including isotope-dilution using a stable isotopically labeled internal standard corresponding to the cleavage product. Quantification is performed based on ratio-based quantification of the cleavage product relative to the labeled internal standard, with optional termination of cleavage and partial purification prior to ionization followed by LC-MS/MS detection using targeted mass spectrometry readouts of multiply charged ions.
Claims Coverage
The claim coverage centers on 2 inventive features.
Exogenous ADAMTS13-cleavable peptide substrate for activity indication
Incubating the sample with an exogenous peptide substrate for ADAMTS13 under conditions allowing enzymatic cleavage to generate an enzymatic cleavage product, wherein the exogenous peptide substrate consists of an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 3 and comprises an ADAMTS13 cleavage site.
Tandem mass spectrometry of ionized enzymatic cleavage product
Ionizing the enzymatic cleavage product to generate a multiply charged gas-phase ion; analyzing the multiply charged gas-phase ion by tandem mass spectrometry to determine presence and/or amount of the enzymatic cleavage product, wherein the presence or amount of the enzymatic cleavage product is indicative of the activity of ADAMTS13 in the sample.
The independent claim coverage centers on converting ADAMTS13 activity into the presence and/or amount of an ADAMTS13-cleavage product from an exogenous peptide substrate, then ionizing that product and using tandem mass spectrometry to detect and/or quantify it as an activity indicator.
Stated Advantages
Higher sensitivity, including a lower limit of quantification (LLOQ) of about 2%.
Specificity.
Throughput and cost reductions versus prior immunoassay/FRET/SELDI-TOF methods.
Documented Applications
Clinical interpretation of ADAMTS13 activity for diagnosis of thrombotic thrombocytopenic purpura (TTP), including that percent normal activity values can be indicative (e.g., <10% indicative of TTP).
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