"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome

Akul Naik; Sanjeeva Srivastava; Arun P. Wiita

doi:10.3791/64952

細胞表面プロテオームのラベルフリー定量のための「Cell Surface Capture」ワークフロー

JoVE Journal
Biochemistry

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JoVE Journal Biochemistry

“Cell Surface Capture” Workflow for Label-Free Quantification of the Cell Surface Proteome

細胞表面プロテオームのラベルフリー定量のための「Cell Surface Capture」ワークフロー

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06:31 min

March 24, 2023

DOI: 10.3791/64952-v

Akul Naik¹, Sanjeeva Srivastava^1,2, Arun P. Wiita^1,3,4

¹Department of Laboratory Medicine,University of California, San Francisco, ²Department of Biosciences and Bioengineering,Indian Institute of Technology Bombay, ³Deptartment of Bioengineering and Therapeutic Sciences,University of California, San Francisco, ⁴Chan Zuckerberg Biohub

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Overview

This protocol describes a workflow for characterizing the cell surface proteome of various cell types, focusing on antigen identification. It includes steps for protein enrichment, sample preparation, and mass spectrometry analysis.

Key Study Components

Area of Science

Proteomics
Cell Biology
Cancer Research

Background

Understanding cell surface proteins is crucial for identifying specific antigens.
Cell membrane proteins can serve as targets for immunotherapies.
Optimizing sample input is essential for accurate results.
This protocol can be applied to various cell types, including tumor cells.

Purpose of Study

To enrich and analyze cell surface proteins from whole cell lysates.
To identify antigens specific to different cell types.
To explore potential targets for cancer immunotherapy.

Methods Used

Cell surface protein enrichment techniques.
Sample preparation for mass spectrometry.
LC-MS/MS analysis for protein identification.
Data processing with specialized software.

Main Results

Successful enrichment of cell membrane proteins.
Identification of unique antigens on tumor cells.
Demonstration of the protocol's effectiveness through iterative optimization.
Potential for discovering novel cancer immunotherapy targets.

Conclusions

The protocol provides a reliable method for studying cell surface proteomes.
It highlights the importance of optimizing experimental conditions.
Findings could lead to advancements in cancer treatment strategies.

Frequently Asked Questions

What is the main goal of this protocol?

The main goal is to characterize the cell surface proteome and identify specific antigens on various cell types.

How can this protocol be applied in cancer research?

It can be used to identify antigens on tumor cells that may serve as targets for immunotherapies.

What techniques are used in this workflow?

The workflow includes cell surface protein enrichment, mass spectrometry analysis, and data processing.

Why is sample optimization important?

Optimizing sample input is crucial for achieving accurate and reliable results in proteomic analysis.

Who demonstrates the procedure in the video?

The procedure is demonstrated by Akul Naik, a junior specialist from the laboratory.

ここでは、さまざまな細胞タイプの細胞表面プロテオームの特性評価のためのプロテオミクスワークフローについて説明します。このワークフローには、細胞表面タンパク質の濃縮、その後のサンプル調製、LC-MS/MSプラットフォームを使用した分析、および専用ソフトウェアによるデータ解析が含まれます。

このプロトコルにより、さまざまな細胞タイプの細胞表面に存在する抗原を調査し、そのタイプの細胞または組織に特異的な抗原を見つけることができます。ここで紹介する技術は、全細胞ライセートから細胞膜タンパク質を濃縮し、最終的には質量分析ベースのプロテオミクスによる分析を可能にします。したがって、このプロトコルの具体的な用途の1つは、腫瘍細胞に適用して、がん免疫療法の新規標的として役立つ可能性のある正常細胞ではなく、それらのがん細胞の表面に存在する抗原を探すことです。

このプロトコルについて注意すべき点の1つは、目的の実験に対してサンプルインプットを最適化することが重要になる場合があるということです。そのため、最適な条件を見つけるために数回の反復または滴定が必要になる場合があります。そこで、この手順を実演するのは、私たちの研究室のジュニアスペシャリストであるAkul Naikです。

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