Multiple-Enzyme-Digestion Strategy Improves Accuracy and Sensitivity of Label- and Standard-Free Absolute Quantification to a Level That Is Achievable by Analysis with Stable Isotope-Labeled Standard Spiking

Quantification
of individual proteins is an essential task in understanding
biological processes. For example, determination of concentrations
of proteins transporting and metabolizing xenobiotics is a prerequisite
for drug disposition predictions in humans based on in vitro data.
So far, this task has frequently been accomplished by targeted proteomics.
This type of analyses requires preparation of stable isotope labeled
standards for each protein of interest. The selection of appropriate
standard peptides is usually tedious and the number of proteins that
can be studied in a single experiment by these approaches is limited.
In addition, incomplete digestion of proteins often affects the accuracy
of the quantification. To circumvent these constrains in proteomic
protein quantification, label- and standard-free approaches, such
as “total protein approach” (TPA) have been proposed.
Here we directly compare an approach using stable isotope labeled
(SIL) standards and TPA for quantification of transporters and enzymes
in human liver samples within the same LC-MS/MS runs. We show that
TPA is a convenient alternative to SIL-based methods. Optimization
of the sample preparation beyond commonly used single tryptic digestion,
by adding consecutive cleavage steps, improves accuracy and reproducibility
of the TPA method to a level, which is achievable by analysis using
stable isotope-labeled standard spiking.