APEX Peroxidase-Catalyzed Proximity Labeling and Multiplexed Quantitative Proteomics

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2008; p. 41
• Main Author: Kalocsay, Marian
• Format: Journal Article
• Language: English
• Published: United States 01.01.2019
• Subjects: Animals; Ascorbate Peroxidases - chemistry; Biotin - analogs & derivatives; Biotin - chemistry; Biotinylation; Cell Line; Humans; Hydrogen Peroxide - chemistry; Mass Spectrometry - methods; Plant Proteins - chemistry; Proteomics - methods; Staining and Labeling - methods; Tyramine - analogs & derivatives; Tyramine - chemistry; Peroxidase catalyzed; Alkaline reversed-phase peptide fractionation; Biotin-tyramide; APEX2; SPS MS3; Quantitative mass spectrometry; Denaturing purification; H2O2; Biotin-phenol; On-bead digest; Streptavidin; Isobaric labeling; TMT; APEX; Proximity labeling
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-9537-0_4

Peroxidase-catalyzed proximity labeling is a powerful technique for defining the molecular environment of proteins in vivo. Expressing a protein of interest fused to a modified plant peroxidase (APEX2) allows labeling of nearby polypeptides. Addition of hydrogen peroxide (H O ) and biotin-tyramide (biotin-phenol) generates short-lived radicals around the peroxidase. Labeling is thus restricted to proteins in close proximity, providing a snapshot of the local environment around the APEX2 fusion protein. Combined with an initial perturbation, progressive changes in interaction partners can be tracked, e.g., after drug treatment. Multiplexed quantitative mass spectrometry permits the parallel analysis of several experimental replicates or of up to 11 time points. Here we describe the denaturing purification of biotin-labeled proteins with magnetic streptavidin beads, and subsequent sample preparation for multiplexed quantitative mass spectrometry. Proximity-labeled proteins are enriched under strong denaturing conditions. Tryptic on-bead digest of purified proteins is combined with tandem mass tag peptide labeling (TMT), alkaline reversed-phase peptide fractionation, and SPS MS mass spectrometry. This analysis pipeline enables studies of complex protein environment changes in perturbed biological systems, as well as comparative studies of functional protein proximity in different cell lines. Through multiplexing, hundreds of proteins can be quantified in each experimental condition in parallel.