Quantification using Signature Hydrolytic Peptides

Labeling with 2H, 13C, 15N and/or lsO can be introduced via peptide synthesis, cell culture, or hydrolysis in labeled water [88]. The heavy isotope-labeled peptide can be used as an IS to obtain quantitative measurements of the protein concentration. Typically, the protein sample of interest is digested with trypsin, and the isotope-labeled control peptides are added to the mixture. The signature peptides in the digest can be separated and quantified by HPLC-ESI-MS/MS. Alternatively, MALDI-MS can also be used for tryptic peptide determinations after some separation steps such as gel electrophoresis [89]. 

The uniqueness of the peptide chosen must be taken into account. Metabolites of the protein drug may have the same sequence, or share a common sequence of the product ions being monitored in the MRM-LC/MS method, possibly resulting in interference. Databases are available to search for tryptic peptide sequences of 

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