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Publication Number
US-10976321-B2
Publication Date
2021-04-13
Expiration Date
Abstract
Provided is a method of assaying for an analyte, including combining a test sample having the analyte, with a calibration sample having at least two different aliquots of the analyte, each aliquot having a different known quantity of the analyte. The test sample and each aliquot are differentially labeled with one or more isobaric mass labels each with a mass spectrometrically distinct mass marker group, such that the test sample and each aliquot of the calibration sample can be distinguished by mass spectrometry. The method further includes determining by mass spectrometry the quantity of analyte in the test sample and in each aliquot, and calibrating the quantity of analyte in the test sample against known and determined quantities of analytes in the aliquots.
Core Innovation
The invention provides a method of assaying for an analyte using covalently isobarically labelled test and calibration samples for mass-spectrometric quantitation. The test sample comprising the analyte is covalently labelled with one or more isobaric mass labels to provide a labelled test sample, and a calibration sample is covalently labelled with two or more isobaric mass labels, where the calibration sample includes at least two different aliquots of the analyte, each aliquot having a different known quantity of the analyte.
The test sample and each analyte aliquot of the calibration sample are differentially labelled with one or more isobaric mass labels, each having a mass spectrometrically distinct mass marker group. After combining the labelled test sample with the labelled calibration sample, the quantity of the analyte in the test sample and the quantity of the analyte in each analyte aliquot of the calibration sample are determined by mass spectrometry, and the quantity in the test sample is calibrated against the known and determined quantities in the aliquots of the calibration sample.
The approach is implemented using isobaric mass labels that include a mass marker group and a mass normalization moiety, with a cleavable linker for forming fragment ions corresponding to the mass marker group. The labels are configured so that the test sample and each calibration aliquot can be distinguished by mass spectrometry, and dissociation or cleavage conditions include collision induced dissociation (CID), electron transfer dissociation (ETD), electron capture dissociation (ECD), and surface induced dissociation (SID).
Claims Coverage
The independent claim is clm-00001. The inventive features are centered on covalent isobaric mass-label differential labelling of a test sample and a multi-aliquot calibration sample, followed by combining and mass-spectrometric determination and calibration against known calibration aliquot quantities.
Differentially label test and calibration aliquots with isobaric markers
Covalently label a test sample comprising the analyte with one or more isobaric mass labels, and covalently label a calibration sample comprising at least two different aliquots of the analyte with different known quantities, wherein the test sample and each analyte aliquot of the calibration sample are differentially labelled with one or more isobaric mass labels, each with a mass spectrometrically distinct mass marker group such that the test sample and each analyte aliquot can be distinguished by mass spectrometry.
Combine labelled test and calibration samples and calibrate against aliquot quantities
Combine the labelled test sample with the labelled calibration sample, determine by mass spectrometry the quantity of the analyte in the test sample and the quantity of the analyte in each analyte aliquot in the calibration sample, and calibrate the quantity of the analyte in the test sample against the known and determined quantities of the analytes in the aliquots in the calibration sample.
Claim clm-00001 covers an assay workflow where covalent isobaric mass labels with mass marker groups enable mass-spectrometric distinction between the labelled test sample and multiple calibration aliquots, and the measured calibration aliquot quantities are used to calibrate the analyte quantity in the test sample.
Stated Advantages
Enables relative and/or absolute quantitation via MS/MS calibration against multiple calibration aliquots using a multi-point standard curve concept without increasing MS complexity.
Documented Applications
Calibration concepts using a known quantitative reference that can be defined by an expected range for a subject state, with determining a percentage change in analyte amount in a test sample.
Use in sample contexts involving plants or animals, where the animal is a human.
Use of a trigger aliquot concept to initiate tandem mass spectrometry scanning during scanning or non-scanning tandem mass spectrometry.
Relative proteome calibration and qualitative reference standard calibration are described.
Low-abundance detection with recombinant reference material is described.
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