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

Summary

Here, we describe a proteomics workflow for characterization of the cell surface proteome of various cell types. This workflow includes cell surface protein enrichment, subsequent sample preparation, analysis using an LC-MS/MS platform, and data processing with specialized software.

Abstract

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of applications. The cell surface proteome (“surfaceome”) in human disease is of significant interest, as plasma membrane proteins are the primary target of most clinically approved therapeutics, as well as a key feature by which to diagnostically distinguish diseased cells from healthy tissues. However, focused characterization of membrane and surface proteins of the cell has remained challenging, primarily due to the complexity of cellular lysates, which mask proteins of interest by other high-abundance proteins. To overcome this technical barrier and accurately define the cell surface proteome of various cell types using mass spectrometry proteomics, it is necessary to enrich the cell lysate for cell surface proteins prior to analysis on the mass spectrometer. This paper presents a detailed workflow for labeling cell surface proteins from cancer cells, enriching these proteins out of the cell lysate, and subsequent sample preparation for mass spectrometry analysis.

Introduction

Proteins serve as the fundamental units by which the majority of cellular functions are carried out. Characterizing the structure and function of relevant proteins is an essential step to understand biological processes. Over the past decade, advances in mass spectrometry technology, analysis software, and databases have enabled the accurate detection and measurement of proteins at a proteome-wide scale¹. Mass spectrometry-based proteomics can be utilized in a diverse array of applications, from basic science analysis of biochemical pathways, to identification of novel drug targets in a translational setting, to diagnosis and monitoring of diseases in the clinic². When screening for novel drug targets, characterization of the cell surface proteome is particularly important, with over 65% of currently approved human drugs targeting cell surface proteins³. The field of cancer immunotherapy also wholly relies on cancer-specific cell surface antigens to target and specifically eliminate tumor cells⁴. Mass spectrometry-based proteomics can thus serve as a promising tool to identify new cell surface proteins toward therapeutic interventions.

However, there are several limitations when utilizing conventional proteomics methods to survey tumor cells for novel cell surface protein targets. A primary concern is that surface proteins make up a very small fraction of the total protein molecules in a cell. Therefore, fragments of these proteins are masked by a high abundance of intracellular proteins when performing mass spectrometry analysis of the whole-cell lysate⁵. This limitation makes it challenging to accurately characterize the cell surface proteome with a traditional proteomics workflow. To address this challenge, it is necessary to develop ways to enrich cell surface proteins out of the whole-cell lysate, prior to analysis on the mass spectrometer. One such method involves the oxidation and biotin labeling of glycosylated cell surface proteins in the intact cells, and subsequent enrichment of these biotinylated proteins from the lysate with a neutravidin pulldown, a process that has been termed "cell surface capture"⁶. Since ~85% of mammalian cell surface proteins are thought to be glycosylated⁷, this serves as an effective method of enriching the cell surface proteome out of the whole cell lysate. This paper describes a complete workflow, beginning with cultured cells, of cell surface biotin labeling, and subsequent sample preparation for mass spectrometry analysis (Figure 1). Over several replicates, this method provides robust coverage of the cell surface proteome of a particular sample. Utilizing this method to characterize the cell surface proteome of both tumor and healthy cells can facilitate the discovery of novel cell surface antigens to identify potential immunotherapeutic targets⁸.

Protocol

NOTE: AMO1 plasmacytoma cells were used for this cell surface proteome experiment. The same protocol could be used for other cell types as well, including a wide array of suspension and adherent cell lines9, as well as various types of primary samples10. However, cell numbers (starting material for the experiment) typically have to be optimized for equivalent proteome coverage. For details related to materials and equipment, see the Table of Materials. For …

Representative Results

For this experiment, we characterized the cell surface proteome of a tumor cell line by labeling N-glycosylated membrane proteins of intact cells with biotin, and enriching these labeled proteins from the whole cell lysate with a neutravidin pulldown (Figure 1). Further, we performed proteome analysis using LC-MS/MS to characterize enriched cell surface proteins. Unlike whole cell proteome analysis, here, the objective was to characterize only cell surface proteins. Hence, we starte…

Discussion

Mass spectrometry-based proteomics is a powerful tool that has enabled unbiased characterization of thousands of unknown proteins on a previously impossible scale. This approach allows us to identify and quantify the proteins, as well as glean a range of insights for the structural and signaling capacities of cells and tissues, by characterizing the variety of proteins present in a particular sample. Moving beyond global protein profiling in a sample, mass spectrometry allows us to characterize various post-translational…

Materials

Kits
96X iST Sample Preparation Kit	PreOmics	P.O.00027	Proteomics sample preparation kit. Includes reagents for reduction, alkylation, and digestion. Also include desalting columns and reagents.
Pierce Quantitative Colorimetric Peptide Assay	Thermo	23275	Peptide quantification kit. Includes peptide standards and components of working reagents.
Reagents
Acetonitrile	Fisher	A955-1
Ammonium bicarbonate	Millipore Sigma	09830-1KG
Biocytin hydrazide	Biotium	90060
D-PBS (w/o Calcium and Magnesium Salts)	UCSF Cell Culture Facility	CCFAL003-225B01
Formic Acid	Honeywell	94318
Halt Protease and Phosphatase Inhibitor Single-Use Cocktail	Thermo	1861280
High Capacity Neutravidin Agarose Resin	Thermo	29204
Phosphate Buffered Saline	UCSF Cell Culture Facility	CCFAL001-22J01
RIPA Lysis Buffer, 10x	Millipore Sigma	20-188
Sodium chloride	Fisher	BP358-212
Sodium metaperiodate	Alfa Aesar	13798
Trypan Blue Stain (0.4%)	Gibco	15250-061
Ultrapure 0.5 M EDTA, pH 8.0	Invitrogen	15575-038
Urea (Proteomics Grade)	VWR	M123-1KG
Equipment
TC20 Automated Cell Counter	Bio-Rad	1450102
PrismR Microcentrifuge	Labnet International	C2500-R-230V
Sonicator	VWR	Branson Sonifier 240
Vacuum Manifold	Promega	Promega Vac-Man
Shaking Heatblock	Eppendorf	Eppendorf Thermomixer C
End-to-End rotator	Labnet	Revolver Adjustable Rotator
LC	Thermo	Ultimate 3000 HPLC and UHPLC
Q Exactive Plus Hybrid Quadrapole Orbitrap Mass Spectrometer	Thermo	IQLAAEGAAPFALGMBDK
Microplate Reader	Biotek	Biotek Synergy 2
Vacuum Concentrator	Labconco	7810010
Supplies
1.5 mL Protein LoBind Tubes	Eppendorf	22431081
1.7 mL Microcentrifuge Tubes
Filtration Columns	Bio-Rad	7326008
Spin Columns	Thermo	69725