Identification of Proteomic Biomarkers of Acetaminophen-Induced Hepatotoxicity Using Stable Isotope Labeling.

Quantitative proteomics approaches based on stable isotopic labeling and mass spectrometry have been widely applied to disease research, drug target discovery, biomarker identification, and systems biology. One of the notable stable isotopic labeling approaches is trypsin-catalyzed O/O labeling, which has its own advantages of low sample consumption, simple labeling procedure, cost-effectiveness, and absence of chemical reactions that potentially generate by-products. In this chapter, a protocol for O/O labeling-based quantitative proteomics approach is described with an application to the identification of proteomic biomarkers of acetaminophen (APAP)-induced hepatotoxicity in rats. The protocol involves first the extraction of proteins from liver tissues of control and APAP-treated rats and digestion into peptides by trypsin. After cleaning of the peptides by solid-phase extraction, equal amounts of peptides from the APAP treatment and the control groups are then subject to trypsin-catalyzed O/O labeling. The labeled peptides are combined and fractionated by off-line strong cation exchange liquid chromatography (SCXLC), and each fraction is then analyzed by nanoflow reversed-phase LC coupled online with tandem mass spectrometry (RPLC-MS/MS) for identification and quantification of differential protein expression between APAP-treated rats and controls. The protocol is applicable to quantitative proteomic analysis for a variety of biological samples.