Parallel Enzyme Digestion for Protein Biomarker Detection

A method for quantifying a polypeptide comprising:

• providing a sample having a polypeptide of interest to be quantified;
• providing a copy of the polypeptide of interest, the copy comprising two amino acids with isotopes, wherein the two amino acids are two lysine, or two arginine, or a lysine and arginine;
• separating the sample into a first sample and a second sample;
• introducing a known quantity of the copy into the first sample and the second sample;
• wherein the copy includes an N-terminal polypeptide linked to a first lysine or a first arginine comprising a first isotope following by a center polypeptide linked to a second lysine or second arginine comprising a second isotope followed by a C-terminal polypeptide;
• introducing an enzyme capable of N-terminal cleavage of lysine or arginine into the first sample, providing a N-terminally cleaved peptide with an N-terminal isotopic lysing or arginine;
• introducing an enzyme cable of C-terminal cleavage of lysine or arginine into the second sample, providing a C-terminally cleaved peptide with a C-terminal isotopic lysing or arginine;
• analyzing the first sample and second sample by mass spectrometry;
• comparing obtained mass spectrometry peak pairs resulting from the N-terminally cleaved peptide of the peptide of interest in the first sample, and N-terminally cleaved peptide of the copy to determine a quantity of the peptide of interest in the sample, providing a first quantity of the peptide of interest;
• and
• comparing obtained mass spectrometry peak pairs resulting from the C-terminally cleaved peptide of the peptide of interest in the second sample, and C-terminally cleaved peptide of the copy to determine a quantity of the peptide of interest in the sample, providing a second quantity of the peptide of interest.