Name: "cell surface capture" workflow for label-free quantification of the cell surface proteome
Uploaded: 2023-03-24T14:20:00
Description: Watch this Scientific Journal Video about "Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome at JoVE.com

We thank Dr. Kamal Mandal (Dept of Laboratory Medicine, UCSF) for help with setting up the LC-MS/MS run, Deeptarup Biswas (BSBE, IIT Bombay) for help with data analysis, and Dr. Audrey Reeves (Dept of Laboratory Medicine, UCSF) for help with data analysis. Related work in the A.P.W. lab is supported by NIH R01 CA226851 and the Chan Zuckerberg Biohub. Figure 1 and Figure 2B were made using BioRender.com.

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 ...

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A., Gomes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+glycosylation:+A+new+road+for+cancer+drug+discovery.">Targeting glycosylation: A new road for cancer drug discovery.</a> Trends in Cancer. 6 (9), 757-766 (2020).</li><li>Gundry, R. L., Boheler, K. R., Van Eyk, J. E., Wollscheid, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+proteomics+in+the+discovery+of+cell-surface+markers+on+stem+cells:+Scratching+the+surface.">A novel role for proteomics in the discovery of cell-surface markers on stem cells: Scratching the surface.</a> Proteomics. Clinical Applications. 2 (6), 892-903 (2008).</li><li>Itzhak, D. 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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-surface+tethered+promiscuous+biotinylators+enable+comparative+small-scale+surface+proteomic+analysis+of+human+extracellular+vesicles+and+cells.">Cell-surface tethered promiscuous biotinylators enable comparative small-scale surface proteomic analysis of human extracellular vesicles and cells.</a> eLife. 11, 73982 (2022).</li><li>Wojtkiewicz, M., Berg Luecke, L., Kelly, M. I., Gundry, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+preparation+of+peptides+for+mass+spectrometry+analysis+in+bottom-up+proteomics+workflows.">Facile preparation of peptides for mass spectrometry analysis in bottom-up proteomics workflows.</a> Current Protocols. 1 (3), 85 (2021).</li><li>V&#228;likangas, T., Suomi, T., Elo, L. 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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...

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 &#34;cell surface capture&#34;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Kits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96X iST Sample Preparation Kit</td>
			<td>PreOmics</td>
			<td>P.O.00027</td>
			<td>Proteomics sample preparation kit. Includes reagents for reduction, alkylation, and digestion. Also include desalting columns and reagents.&#160;</td>
		</tr>
		<tr>
			<td>Pierce Quantitative Colorimetric Peptide Assay</td>
			<td>Thermo</td>
			<td>23275</td>
			<td>Peptide quantification kit. Includes peptide standards and components of working reagents.&#160;</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher</td>
			<td>A955-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Millipore Sigma</td>
			<td>09830-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocytin hydrazide</td>
			<td>Biotium</td>
			<td>90060</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS (w/o Calcium and Magnesium Salts)</td>
			<td>UCSF Cell Culture Facility</td>
			<td>CCFAL003-225B01</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Honeywell</td>
			<td>94318</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Single-Use Cocktail</td>
			<td>Thermo</td>
			<td>1861280</td>
			<td></td>
		</tr>
		<tr>
			<td>High Capacity Neutravidin Agarose Resin</td>
			<td>Thermo</td>
			<td>29204</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>UCSF Cell Culture Facility</td>
			<td>CCFAL001-22J01</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA Lysis Buffer, 10x</td>
			<td>Millipore Sigma</td>
			<td>20-188</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium metaperiodate</td>
			<td>Alfa Aesar</td>
			<td>13798</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure 0.5 M EDTA, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea (Proteomics Grade)</td>
			<td>VWR</td>
			<td>M123-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>Bio-Rad</td>
			<td>1450102</td>
			<td></td>
		</tr>
		<tr>
			<td>PrismR Microcentrifuge</td>
			<td>Labnet International</td>
			<td>C2500-R-230V</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>VWR</td>
			<td>Branson Sonifier 240</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Manifold</td>
			<td>Promega</td>
			<td>Promega Vac-Man</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking Heatblock</td>
			<td>Eppendorf</td>
			<td>Eppendorf Thermomixer C</td>
			<td></td>
		</tr>
		<tr>
			<td>End-to-End rotator</td>
			<td>Labnet</td>
			<td>Revolver Adjustable Rotator</td>
			<td></td>
		</tr>
		<tr>
			<td>LC</td>
			<td>Thermo</td>
			<td>Ultimate 3000 HPLC and UHPLC</td>
			<td></td>
		</tr>
		<tr>
			<td>Q Exactive Plus Hybrid Quadrapole Orbitrap Mass Spectrometer</td>
			<td>Thermo</td>
			<td>IQLAAEGAAPFALGMBDK</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>Biotek</td>
			<td>Biotek Synergy 2&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Concentrator</td>
			<td>Labconco</td>
			<td>7810010</td>
			<td></td>
		</tr>
		<tr>
			<td>Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Protein LoBind Tubes</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 mL Microcentrifuge Tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filtration Columns</td>
			<td>Bio-Rad</td>
			<td>7326008</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Columns</td>
			<td>Thermo</td>
			<td>69725</td>
			<td></td>
		</tr>
	</tbody>
</table>

"cell surface capture" workflow for label-free quantification of the cell surface proteome

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.

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...

Watch this Scientific Journal Video about "Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome at JoVE.com

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

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.

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of ...

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Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

肾小球的分离及肾小球细胞表面蛋白的活体标记

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

一次人气道上皮细胞气道表面液体 pH 值的实时、半自动荧光测量

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

SUMO 结合实体 （SUBEs） 作为肝癌中 SUMO 蛋白酶的浓缩、分离、识别和表征的工具

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

基于无标签阻抗的 GPCR 检测中用于提高吞吐量的逐步加量协议

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

用于植物微RNA检测和定量的RNA斑点分析

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...