Multipurpose mass spectrometric assay panels for peptides
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Publication Number
US-9588126-B2
Publication Date
2017-03-07
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 in 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
A method measures the relative amounts of a first and a second compartment of a biological sample by selecting a first monitor peptide that is proteotypic for a first protein whose amount in the sample is contributed predominantly by the first compartment, and selecting a second monitor peptide that is proteotypic for a second protein whose amount in the sample is contributed predominantly by the second compartment. The sample is digested by a proteolytic process to produce a digest, and labeled versions of the first and second peptides differing from the first and second peptides by a mass increment are added.
The method measures the amounts of the first monitor peptide and the labeled version of the first monitor peptide using a mass spectrometer and divides the measured amount to produce a first ratio, then multiplies the first ratio by the known amount of the labeled version of the first peptide to yield an amount of the first peptide in the digest. In parallel, it measures the amounts of the second monitor peptide and the labeled version of the second monitor peptide, divides to produce a second ratio, and multiplies by the known amount of the labeled version of the second peptide to yield an amount of the second peptide in the digest.
The method calculates the relative amounts of the first and second compartments using the relative amounts of the first and second peptides in the digest together with previous measurements of the concentrations of the first peptide, or the first protein, in the first compartment and of the second peptide, or the second protein, in the second compartment. This supports multipurpose quantitative mass spectrometric estimation of compartment volumes and compositions in mixed samples where compartments are disrupted, including hematocrit estimation and reconstruction of complete blood count/differentials by converting peptide quantities to cell counts using pre-established copy numbers.
Claims Coverage
Two independent claims are present. Both cover measuring relative compartment amounts using proteotypic monitor peptides, proteolytic digestion, mass spectrometric quantification with mass-labeled internal standards, ratio calculations, and compartment computation using prior compartment concentration information; Claim 1 additionally uses known amounts of labeled peptide standards.
Ratio-based compartment quantification using proteotypic monitor peptides and labeled standards
Selecting a first monitor peptide and a second monitor peptide that are proteotypic for proteins whose amounts are contributed predominantly by the first and second compartments; digesting the sample to produce a digest; adding labeled versions of the first and second peptides differing by a mass increment in known amounts; measuring unlabeled and labeled peptides by a mass spectrometer; dividing measured amounts to produce ratios and multiplying ratios by the known amounts of labeled peptides to yield peptide amounts in the digest; and calculating the relative amounts of the first and second compartments using the relative amounts of the first and second peptides together with previous measurements of concentrations of the first peptide, or first protein, in the first compartment and of the second peptide, or second protein, in the second compartment.
Relative compartment quantification using ratio-derived peptide amounts and prior compartment concentrations
Selecting a first monitor peptide and a second monitor peptide that are proteotypic for proteins whose amounts are contributed predominantly by the first and second compartments; digesting the sample to produce a digest; adding labeled versions of the first and second peptides differing by a mass increment in relative amounts; measuring unlabeled and labeled peptides by a mass spectrometer; dividing measured amounts to yield relative amounts of the first and second peptides in the digest; and calculating the relative amounts of the first and second compartments using the relative amounts of the first and second peptides together with the relative amounts of the labeled versions of the first and second monitor peptides and previous measurements of concentrations of the first peptide, or first protein, in the first compartment and of the second peptide, or second protein, in the second compartment.
Across the independent claims, compartment estimation is driven by proteotypic monitor peptides associated with proteins predominantly contributed by each compartment, proteolytic digestion, mass spectrometric measurement of unlabeled versus mass-increment labeled peptide versions, ratio-based derivation of peptide abundances, and computation of relative compartment amounts using those peptide-derived abundances together with prior compartment concentration information.
Stated Advantages
Enables quantitative measurement of the relative amounts of first and second compartments in a biological sample using a multipurpose quantitative mass spectrometric approach.
Supports estimating relative compartment volumes and compositions in mixed samples where compartments are disrupted, including hematocrit determination and reconstruction of complete blood count/differentials.
Provides hematocrit-driven correction to protein analytes to reduce variability after hematocrit correction.
Documented Applications
Hematocrit estimation from dried blood by measuring plasma proteins and red blood cell markers and correcting protein measurements for hematocrit-driven plasma/RBC dilution.
Reconstructing complete blood counts/differentials by measuring cell-type compartment-specific monitor peptides and converting peptide quantities to cell counts using pre-established copy numbers.
Estimating total blood volume in tissues.
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