Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Target Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Query: EC:2.4.1.14 (
SPS
)
813
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
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
2
O
2
) 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
3
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.
...
PMID:APEX Peroxidase-Catalyzed Proximity Labeling and Multiplexed Quantitative Proteomics. 3112 87
