Identification of Human Liver Microsomal Proteins Adducted by a Reactive Metabolite Using Shotgun Proteomics

Covalent modification of cellular
proteins by chemically reactive
compounds/metabolites has the potential to disrupt biological function
and elicit serious adverse drug reactions. Information on the nature
and binding patterns of protein targets are critical toward understanding
the mechanism of drug induced toxicity. Protein covalent binding studies
established in liver microsomes can quantitively estimate the extent
of protein modification, but they provide little information on the
nature of the modified proteins. In this article, we describe a label-free
shotgun proteomic workflow for the identification of target proteins
modified in situ by reactive metabolites in human liver microsome
incubations. First, we developed a shotgun proteomic workflow for
the characterization of the human liver microsomal subproteome, which
consists of predominately membrane-bound proteins. Human liver microsomes
were solubilized with a combination of MS-compatible organic solvents
followed by protein reduction, alkylation, and tryptic digestion.
The unmodified samples were analyzed by UHPLC-MS/MS, and the proteins
were identified by database searching. This workflow led to the successful
identification of 329 human liver microsomal subproteome proteins
with 1% FDR (false discovery rate). The same method was then applied
to identify the modifications of human liver microsomal proteins by
a known reactive metabolite 2-(methylsulfonyl)­benzo­[d]­thiazole (2), either after incubation directly with 2 or with its parent compound 2-(methylthio)­benzo­[d]­thiazole (1). A total of 19 modified constituent
peptides which could be mapped to 18 proteins were identified in human
liver microsomes incubated directly with 2. Among these,
5 modified constituent peptides which could be mapped to 4 proteins
were identified in incubation with 1, which is known
to generate 2 in human liver microsomal incubations.
This label-free workflow is generally applicable to the identification
and characterization of proteins adducted with reactive metabolites
in complex matrices and may serve as a valuable tool to understand
the link between protein targets and clinically relevant toxicities.