Patent ID: 8871688

Claim:
A method for quantifying a target polypeptide in a sample comprising the steps of: (a) providing a sample containing a full length target polypeptide to be quantified; (b) adding a known quantity of an in vitro synthesized, isotope labeled full length homolog of said full length target polypeptide wherein the homolog has the identical amino acid sequence as the full length target polypeptide to be quantified except that all lysine residues in said homolog are labeled with 13 C and/or 15 N such that all the lysine residues in said homolog are [ 13 C and/or 15 N]-lysine and all arginine residues in said homolog are labeled with 13 C and/or 15 N such that all the arginine residues in said homolog are [ 15 N and/or 13 C]-arginine thereby generating a spiked sample comprising the polypeptide to be quantified and the isotope labeled homolog; (c) treating the spiked sample with trypsin to digest the polypeptide and the isotope labeled homolog in said spiked sample to generate a plurality of proteolytic peptides; (d) analysing the proteolytic peptides generated in step c) by mass spectrometry (MS); (e) determining a ratio of an isotope-labeled proteolytic peptide to the corresponding unlabeled proteolytic peptide; and (f) calculating from the ratio and the known quantity of the isotope-labeled target polypeptide, the quantity of the target polypeptide in the sample.