Methods for measuring concentrations of biomolecules

A method of determining the absolute concentration of an amyloid-beta protein (Aβ), isoform, variant or digestion product thereof in a subject, comprising:

• (a) metabolically labeling the endogenous Aβ protein isoform, variant or digestion product thereof by administering at least one labeled moiety to the subject, wherein the at least one labeled moiety metabolically incorporates into the endogenous Aβ protein isoform, variant or digestion product thereof;
• (b) contacting a biological sample obtained from the subject with a Quantitation Standard (QS) comprising a known concentration of a biomolecule labeled with two labeled moieties such that the labeled QS has a molecular weight that differs from the metabolically labeled endogenous Aβ protein isoform, variant or digestion product thereof, wherein the QS is an Aβ peptide, wherein the biological sample comprises a metabolically labeled fraction of the endogenous Aβ protein isoform, variant or digestion product thereof and an unlabeled fraction of the Aβ protein, isoform, variant or digestion product thereof;
• (c) isolating the metabolically labeled, the unlabeled and the labeled QS forms of Aβ protein isoform, variant or digestion product thereof from the biological sample using a detergent to increase the labeled Aβ, unlabeled Aβ peptides and QS solubility;
• (d) maintaining the labeled Aβ, unlabeled Aβ peptides and QS in a reduced state to increase said peptides detection;
• (e) subjecting the labeled Aβ, unlabeled Aβ peptides and QS to mass spectroscopy analysis;
• and
• (f) calculating the absolute concentration of the endogenous, metabolically labeled and unlabeled, Aβ protein isoform, variant or digestion product thereof using the known concentration of Aβ protein isoform, variant or digestion product in the labeled QS.