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Methods Mol Biol DOI:10.1007/978-1-4939-9124-2_18

In Situ Peroxidase Labeling and Mass Spectrometry of Alpha-Synuclein in Rat Cortical Neurons.

Publication TypeJournal Article
Year of Publication2019
AuthorsSanz, R, Ovando-Roche, P, Udeshi, ND, Carr, SA, Chung, CYeun, Khurana, V
JournalMethods Mol Biol
Volume1948
Pages235-246
Date Published2019
ISSN1940-6029
Abstract

In this chapter, we describe a novel ascorbate peroxidase (APEX)-based labeling method that in combination with mass spectrometry identifies proteins in the immediate vicinity of αSyn in living rat cortical neurons. To isolate these interactions, we transduced primary cortical neurons with a lentivirus encoding APEX2 tagged to the C-terminus of alpha-synuclein (αSyn) and under the control of a synapsin promoter. Neural protein lysates were then incubated with streptavidin magnetic beads, washed, eluted from the beads, and digested overnight. The desalted peptides were then labeled with iTRAQ (4-plex) reagents and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (LC-MS/MS). Collected data were analyzed using Spectrum Mill software, ultimately shedding light on αSyn physiological function and abnormal behavior during pathology.

DOI10.1007/978-1-4939-9124-2_18
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/30771182?dopt=Abstract

Alternate JournalMethods Mol. Biol.
PubMed ID30771182