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-11959124-B2
Publication Date
2024-04-16
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 invention provides methods and systems for determining ADAMTS13 enzyme activity in a sample. A sample is incubated with an exogenous peptide substrate that comprises an ADAMTS13 cleavage site, such that ADAMTS13 enzymatically cleaves the substrate to produce an enzymatic cleavage product. The enzymatic cleavage product comprises a sequence with at least 70% sequence identity to SEQ ID NO:4, and the cleavage product sequence is used as the readout for ADAMTS13 activity.
After cleavage, the enzymatic cleavage product is ionized to generate a multiply charged gas-phase ion. The presence or amount of the enzymatic cleavage product in the sample is determined by analyzing the multiply charged gas-phase ion by tandem mass spectrometry, including LC-MS/MS, to indicate the presence or amount of ADAMTS13 enzyme activity in the sample. The approach quantifies the cleavage product by measuring a cleavage product signal in tandem mass spectrometry and can report enzyme activity measures such as specific activity and percent normal activity.
In some embodiments, stable isotopically labeled internal standards are used to support quantification, and the ratio of enzymatic cleavage product to internal standard is reported as a measure of enzyme activity. The document describes optional handling steps such as partial purification and reaction termination prior to tandem mass spectrometry, with sample types including plasma and serum. Systems are described as including stations for incubation, ionization, mass spectrometry analysis, and optional chromatography or partial purification, with additional optional features for sample cleanup and enrichment.
Claims Coverage
The independent claim is directed to a method for determining ADAMTS13 enzyme activity by detecting an ADAMTS13-specific enzymatic cleavage product using ionization and tandem mass spectrometry. The claim set supports eight inventive features, including substrate incubation, ionization, tandem mass spectrometry detection, sequence identity constraints, specified ionization techniques, targeted m/z analysis, and isotopically labeled internal standards.
Incubating with an ADAMTS13-cleavable exogenous peptide substrate
Incubating the sample with an exogenous peptide substrate comprising an ADAMTS13 cleavage site under conditions allowing enzymatic cleavage by ADAMTS13 to produce an enzymatic cleavage product, wherein the enzymatic cleavage product comprises a sequence with at least 70% sequence identity to SEQ ID NO:4.
Ionizing the cleavage product to generate multiply charged gas-phase ions
Ionizing the enzymatic cleavage product to generate a multiply charged gas-phase ion of the enzymatic cleavage product.
Tandem mass spectrometry detection of cleavage product as indicator of activity
Analyzing the multiply charged gas-phase ion by tandem mass spectrometry to determine presence or amount of enzymatic cleavage product in the sample, wherein the presence or the amount of the enzymatic cleavage product in the sample is indicative of presence or amount of enzyme activity of ADAMTS13 in the sample.
Sequencing identity anchored to SEQ ID NO:4
The method further defines that the enzymatic cleavage product comprises SEQ ID NO:4.
Higher sequence identity for the exogenous peptide substrate
The method uses an exogenous peptide substrate that has at least 90% sequence identity to SEQ ID NO:3.
Specified ionization techniques
The method specifies that step (b) ionizes the enzymatic cleavage product using an ionization technique selected from electrospray ionization, atmospheric pressure chemical ionization, and atmospheric pressure photoionization.
Targeted m/z value selection for tandem MS analysis
The method specifies that step (c) uses ions with m/z values including at least one of the listed ratios.
Isotopically labeled internal standard co-measurement
The method further determines the presence or amount of an isotopically labeled internal standard alongside the presence or amount of an enzymatic cleavage product in step (c).
Overall claim coverage centers on using an exogenous ADAMTS13-cleavable peptide substrate to generate an ADAMTS13-specific cleavage product, ionizing that product to multiply charged ions, and detecting the presence or amount of the cleavage product by tandem mass spectrometry as an indicator of ADAMTS13 enzyme activity. Dependent features further anchor the cleavage product and substrate sequences to SEQ ID sequences, constrain ionization and targeted m/z analysis, and optionally incorporate isotopically labeled internal standards for quantification.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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