Multipurpose mass spectrometric assay panels for peptides
Inventors
Assignees
SISCAPA ASSAY TECHNOLOGIES Inc • Siscapa Assay Technologies Invc
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Publication Number
US-10254292-B2
Publication Date
2019-04-09
Expiration Date
Abstract
Methods are provided for estimating the relative amounts of identifiable compartments, such as different types of cells or cell components, within a biological sample. The methods use mass spectrometric analysis to quantitate compartment-specific molecules and thereby allow calculation of the amount of each compartment that is present in a biological sample. The methods can, for example, provide a measurement of hematocrit from a dried blood sample.
Core Innovation
The invention relates to multipurpose quantitative mass-spectrometric assays for multi-compartment biological samples in which compartment-specific molecules are measured and used to quantify cell populations and protein biomarkers. Compartment specific molecules are measured in a proteolytic digest by quantitative mass spectrometry using at least one monitor peptide, with the measured monitor peptide amounts determined by isotope dilution approaches.
A key feature is reconstructing blood compartment volumes and estimating hematocrit from dried blood spots despite compartment mixing, and then correcting protein biomarker measurements for hematocrit variation. This correction is applied to quantified protein biomarkers such as protein C inhibitor (PCI) and soluble transferrin receptor (sTfR) to reduce variability related to hematocrit or packed cell volume.
For multi-compartment blood samples, the invention further uses CSM copy numbers per cell to infer the number of cells constituting each compartment. CSMs and their monitor peptide sets are defined for multiple cell compartments, including leukocyte subsets and platelets, enabling calculation of cell numbers by dividing the measured number of CSM molecules by pre-established average CSM molecule counts per cell in each compartment.
Claims Coverage
The document provides two independent claims directed to measuring the number of cells constituting a compartment in a multi-compartment biological sample and to measuring the number of cells constituting each of a multiplicity of compartments in a blood sample. The core inventive features are quantitative mass spectrometry of compartment-specific molecules using monitor peptides and calculation of cell numbers by dividing measured CSM molecules by pre-established average CSM molecules per cell.
Quantitative CSM measurement in a proteolytic digest using monitor peptides
Measuring the amount of, or the number of molecules of, a compartment specific molecule specific to said cells, wherein said CSM is measured in a proteolytic digest of said sample by quantitative mass spectrometry of at least one monitor peptide.
Cell-number calculation from measured CSM molecules divided by pre-established average CSM molecules per cell
Calculating the number of said cells in said sample by dividing said measured number of molecules by the pre-established average number of CSM molecules occurring in each of said cells of said compartment.
Multi-compartment blood measurement with cell-count calculation for each compartment
Measuring in said sample the amount of, or equivalently the number of molecules of, a compartment-specific molecule specific to cells of each of said compartments, wherein said CSM is measured in a proteolytic digest of said sample by quantitative mass spectrometry of at least one monitor peptide; calculating the number of said cells in each of said compartments of said sample by dividing said measured number of molecules by the respective pre-established average number of CSM molecules occurring in each of said cells of said compartment; wherein said compartments are selected from the group consisting of red blood cells, B lymphocytes, T lymphocytes, helper T lymphocytes, NK cells, monocytes, neutrophils, eosinophils, and platelets.
Across the independent claims, the inventive concept centers on quantifying compartment-specific molecules from a proteolytic digest using quantitative mass spectrometry of monitor peptides, and converting the measured CSM molecule numbers into cell counts by dividing by pre-established average CSM molecule counts per cell. In the blood-sample claim, the compartments are explicitly selected from red blood cells, lymphocyte subsets, NK cells, monocytes, neutrophils, eosinophils, and platelets.
Stated Advantages
Reducing biomarker variability after hematocrit correction for protein biomarkers such as PCI and soluble transferrin receptor.
Documented Applications
Measuring the number of cells constituting a compartment of a multi-compartment biological sample using compartment-specific molecules and quantitative mass spectrometry with monitor peptides.
Measuring the number of cells constituting each of a multiplicity of compartments in a blood sample by quantifying CSM molecules in a proteolytic digest and calculating compartment cell numbers by division using pre-established average CSM molecule counts per cell.
Correcting quantified protein biomarker measurements, including PCI and soluble transferrin receptor, for hematocrit variation when using dried blood spots, and reconstructing plasma/RBC compartment volumes and estimating hematocrit despite compartment mixing.
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