# Protocol for identifying surface membrane proteins and their associated proteome from mouse cortical neuron cultures by in situ biotinylation

## TL;DR

This paper describes a protocol to identify surface proteins on mouse neurons using biotinylation, enabling analysis of membrane proteins under different conditions.

## Contribution

A detailed protocol for in situ biotinylation and mass spectrometry-based profiling of surface membrane proteins in mouse cortical neurons.

## Key findings

- The protocol enables quantitative analysis of membrane-associated proteins in neurons.
- In situ biotinylation allows for labeling and purification of surface proteins in live neurons.

## Abstract

Modifications to the plasma membrane proteome reflect neuronal physiological states. Here, we present a protocol to profile surface proteins from mouse cortical neuron cultures using in situ biotinylation. The workflow includes neuron culture, surface labeling, enrichment of biotinylated proteins, and on-beads digestion followed by mass spectrometry, enabling quantitative and comparative analysis of membrane-associated proteins across diverse physiological conditions.

For complete details on the use and execution of this protocol, please refer to Shi et al.1

•Instructions for labeling membrane proteins in situ in live neurons•Procedures for purifying membrane and membrane-associated proteins•Steps for identifying membrane proteins that report local membrane events

Instructions for labeling membrane proteins in situ in live neurons

Procedures for purifying membrane and membrane-associated proteins

Steps for identifying membrane proteins that report local membrane events

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Modifications to the plasma membrane proteome reflect neuronal physiological states. Here, we present a protocol to profile surface proteins from mouse cortical neuron cultures using in situ biotinylation. The workflow includes neuron culture, surface labeling, enrichment of biotinylated proteins, and on-bead digestion followed by mass spectrometry, enabling quantitative and comparative analysis of membrane-associated proteins across diverse physiological conditions.