Analytical Approach to Obtain Absolute Abundance of Endogenous Proteins using LC-MS/MS, presented at the AAPS National Biotechnology Conference, May 16-18, 2011 in San Francisco, CA.

Purpose: The realization of in vitro/in vivo correlations and ultimately human pharmacokinetic (PK) and drug-drug interaction (DDI) predictions depends on the ability to scale in vitro data generated in ADME model systems to the in vivo situation. One of the most effective means of properly defining these scaling factors is through the incorporation of drug metabolizing enzyme concentrations. However, enzyme concentrations in these model systems are inferred from mRNA measurements or enzyme activity data, and little is typically known about the concentration of proteins.

UGTs are primary drug metabolizing enzymes responsible for glucuronidation of xenobiotics and endogenous metabolites. UGT-catalyzed reactions are a major metabolic pathway in humans. While mRNA and activity data are available for many UGT isoforms in liver microsomal reagents, little or no information is available on the relative or absolute abundance of UGT isoforms.

Absolute quantification necessitates the use of qualified standards of known concentration and purity. In cases where a qualified standard is not available, stable labeled peptides may be used to qualify a recombinant protein \“standard\”, which is subsequently used to build calibrators. This presentation will highlight the analytical approach used to obtain absolute abundance of UGT isoforms in human liver microsomes using LC-MS/MS.

Methods: The analytical approach is divided into 3 parts: 1) qualification of the recombinant protein standard using isotope dilution, 2) use of this qualified standard to build calibrators in a surrogate matrix, and3) quantification of the endogenous protein in the relevant matrix. For LC-MS/MS analysis, the surrogate peptide approach was used, where two unique tryptic peptides for each isozyme were used for quantification. Lower limits of quantification varied with isozyme (0.5 – 5 pmol isozyme/mg of total protein).

Results: Six (6) recombinant human UGT microsomes were qualified as protein standards as described. Using these protein standards, assay precision and accuracy were better than 20% in most determinations. Fifty (50) human liver microsomal donors were assessed in triplicate for abundance of UGT isozymes, with precision less than 20% CV.

Conclusions: The LC-MS/MS analytical approach described may be used to determine the absolute abundance of endogenous proteins when qualified protein standards are not available.


To gain access to full literature please fill out this form.

[contact-form-7 404 "Not Found"]