In Situ Peroxidase Labeling and Mass Spectrometry of Alpha-Synuclein in Rat Cortical Neurons.
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.
['Sanz|Ricardo|R|', 'Ovando-Roche|Patrick|P|', 'Udeshi|Namrata D|ND|', 'Carr|Steven A|SA|', 'Chung|Chee Yeun|CY|', 'Khurana|Vikram|V|']
D000818:Animals D002478:Cells, Cultured D002540:Cerebral Cortex D000078332:Data Analysis D005455:Fluorescent Antibody Technique D006801:Humans D017403:In Situ Hybridization D013058:Mass Spectrometry D009474:Neurons D010300:Parkinson Disease D009195:Peroxidase D040901:Proteomics D051381:Rats D013194:Staining and Labeling D051844:alpha-Synuclein
Mass spectrometry Alpha-synuclein Neurons APEX2 APEX Parkinson’s disease Synucleinopathies