Cell-Type-Specific Protein Metabolic Labeling and Identification Using the Methionine Subrogate ANL in Cells Expressing a Mutant Methionyl-tRNA Synthetase

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2025 Mar 11

PMID 40067620

Authors

Rodrigo Alvarez-Pardo

Ella Doron-Mandel

Hector Albert-Gasco

Cristina Olmedo Salinas

Marko Jovanovic

Beatriz Alvarez-Castelao

Affiliations

Soon will be listed here.

Abstract

The study of protein homeostasis in vivo is crucial for our understanding of the functions of cells and organisms. However, complex organisms, such as mammals, are built from heterogeneous tissues and cell-types. These cell-types are often specialized and react in different ways to the same physiological or pathological stimulus. Therefore, a major challenge in proteomics is the identification of proteomes and their behavior in a cell-type-specific manner. In this protocol, we describe a technique to label, enrich, and identify proteins from specific cell types. This technique is based on the expression of a mutant methionyl-tRNA synthetase (MetRS*) for incorporation of a bioorthogonal analog of methionine (ANL) into proteins. ANL can be subsequently bound to an alkyne by click-chemistry, which is used as a bait for protein purification followed by mass spectrometry identification.

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