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

Ricardo Sanz; Patrick Ovando-Roche; Namrata D Udeshi; Steven A Carr; Chee Yeun Chung; Vikram Khurana

doi:10.1007/978-1-4939-9124-2_18

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

Methods Mol Biol. 2019:1948:235-246. doi: 10.1007/978-1-4939-9124-2_18.

Authors

Ricardo Sanz¹, Patrick Ovando-Roche¹, Namrata D Udeshi², Steven A Carr², Chee Yeun Chung³, Vikram Khurana^{4

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Affiliations

¹ Ann Romney Center for Neurologic Disease, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
² Broad Institute of MIT and Harvard, Cambridge, MA, USA.
³ Yumanity Therapeutics, Cambridge, MA, USA. cychung@yumanity.com.
⁴ Ann Romney Center for Neurologic Disease, Department of Neurology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA. vkhurana@bwh.harvard.edu.
⁵ Broad Institute of MIT and Harvard, Cambridge, MA, USA. vkhurana@bwh.harvard.edu.
⁶ Harvard Stem Cell Institute, Cambridge, MA, USA. vkhurana@bwh.harvard.edu.

PMID: 30771182
DOI: 10.1007/978-1-4939-9124-2_18

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.

Keywords: APEX; APEX2; Alpha-synuclein; Mass spectrometry; Neurons; Parkinson’s disease; Synucleinopathies.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cells, Cultured
Cerebral Cortex / cytology
Cerebral Cortex / metabolism
Data Analysis
Fluorescent Antibody Technique
Humans
In Situ Hybridization*
Mass Spectrometry*
Neurons / metabolism*
Parkinson Disease / metabolism
Peroxidase / metabolism*
Proteomics / methods
Rats
Staining and Labeling
alpha-Synuclein / chemistry*
alpha-Synuclein / metabolism*

Substances

alpha-Synuclein
Peroxidase