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 ...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics:+technologies+and+their+applications.">Proteomics: technologies and their applications.</a> Journal of Chromatographic Science. 55 (2), 182-196 (2017).</li><li>Bausch-Fluck, D., 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=The+in+silico+human+surfaceome.">The in silico human surfaceome.</a> Proceedings of the National Academy of Sciences. 115 (46), 10988-10997 (2018).</li><li>Takahashi, Y., 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=Research+advance+in+tumor+specific+antigens:+A+narrative+review.">Research advance in tumor specific antigens: A narrative review.</a> AME Medical Journal. 6, 35 (2021).</li><li>Li, Y., Qin, H., Ye, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+on+enrichment+methods+for+cell+surface+proteome+profiling.">An overview on enrichment methods for cell surface proteome profiling.</a> Journal of Separation Science. 43 (1), 292-312 (2020).</li><li>Wollscheid, B., 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=Mass-spectrometric+identification+and+relative+quantification+of+N-linked+cell+surface+glycoproteins.">Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins.</a> Nature Biotechnology. 27 (4), 378-386 (2009).</li><li>Chandler, K. 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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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+evaluation+of+popular+proteomics+software+workflows+for+label-free+proteome+quantification+and+imputation.">A comprehensive evaluation of popular proteomics software workflows for label-free proteome quantification and imputation.</a> Briefings in Bioinformatics. 19 (6), 1344-1355 (2018).</li><li>Cox, J., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MaxQuant+enables+high+peptide+identification+rates,+individualized+p.p.b.-range+mass+accuracies+and+proteome-wide+protein+quantification.">MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.</a> Nature Biotechnology. 26 (12), 1367-1372 (2008).</li><li>Bausch-Fluck, D., 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=A+mass+spectrometric-derived+cell+surface+protein+atlas.">A mass spectrometric-derived cell surface protein atlas.</a> PLoS One. 10 (3), 0121314 (2015).</li><li>Griss, J., 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=ReactomeGSA-efficient+multi-omics+comparative+pathway+analysis.">ReactomeGSA-efficient multi-omics comparative pathway analysis.</a> Molecular &amp; Cellular Proteomics. 19 (12), 2115-2125 (2020).</li><li>Waas, M., 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=SurfaceGenie:+a+web-based+application+for+prioritizing+cell-type-specific+marker+candidates.">SurfaceGenie: a web-based application for prioritizing cell-type-specific marker candidates.</a> Bioinformatics. 36 (11), 3447-3456 (2020).</li><li>Mellacheruvu, D., 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=The+CRAPome:+a+contaminant+repository+for+affinity+purification-mass+spectrometry+data.">The CRAPome: a contaminant repository for affinity purification-mass spectrometry data.</a> Nature Methods. 10 (8), 730-736 (2013).</li><li>van Oostrum, M., 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=Classification+of+mouse+B+cell+types+using+surfaceome+proteotype+maps.">Classification of mouse B cell types using surfaceome proteotype maps.</a> Nature Communications. 10 (1), 5734 (2019).</li><li>Berg Luecke, L., Gundry, R. 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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

&quot;Cell Surface Capture&quot; 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.

"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 ...

This protocol allows us to investigate antigens present on the cell surface of various cell types to find antigens specific to that type of cell or tissue. The technique we present here allows for enrichment of cell membrane proteins out of a whole cell lysate, ultimately for analysis by mass spectrometry-based proteomics. So one specific application of this protocol is to apply it to tumor cells to look for antigens present on the surface of those cancer cells and not normal cells that could serve as novel targets for cancer immunotherapies.One thing to note about this protocol is that it can be important to optimize your sample input for your experiment of interest. So it may take several iterations or titrations to find the optimal conditions. So demonstrating the procedure will be Akul Naik, a junior specialist from our laboratory.To begin, remove the frozen biotin-labeled cell pellet from minus 80 degrees Celsius and thaw it on ice. Add 500 microliters of 2X Lysis Buffer to the pellet, mix thoroughly, and vortex vigorously for 30 seconds. Sonicate the cell lysate on ice, and centrifuge the lysate at 17, 200 G for 10 minutes at 4 degrees Celsius to pellet any remaining debris.While the lysate is centrifuging, add 100 microliters of NeutrAvidin Agarose resin beads to a filtration column attached to the vacuum manifold. Apply a gentle vacuum and wash the beads by flowing 3 milliliters of Wash Buffer 1 over them. Remove the filtration column from the vacuum manifold, cap the bottom, and add the clarified cell lysate to the column with the beads.Cap the top of the column and incubate the lysate with the beads on an end-to-end rotor for 120 minutes at 4 degrees Celsius. After completing the lysate incubation, place the filtration column back onto the vacuum manifold, and apply a gentle vacuum. Wash the beads by flowing five milliliters of Wash Buffer 1 over them.Repeat the washing step with five milliliters of Wash Buffer 2 and Wash Buffer 3. Once the final Wash Buffer has flowed entirely through the beads, remove the column from the manifold. Then add 100 microliters of lyse solution to the beads and transfer them to a 1.7-milliliter low protein binding micro centrifuge tube with a pipette.Briefly spin the tube to settle the beads and remove 50 microliters of the solution. Next, place the tube on a heat block shaker at 65 degrees Celsius for 10 minutes at 1000 RPM shaking. Resuspend the dried trypsin in the digest tube from the kit by adding 210 microliters of the resuspend solution and pipetting it up and down several times to ensure all the dried trypsin is resuspended.At the end of bead incubation, remove it from the heat block shaker and add 50 microliters of the resuspended trypsin solution to the beads. Incubate the tube at 37 degrees Celsius, shaking at 500 RPM for at least 90 minutes to digest the protein. Once the digestion is complete, transfer the solution containing the beads to a spin column and insert the column into a 1.7-milliliter low protein binding micro centrifuge tube.Spin the tube to separate the digested peptide solution from the beads. Next, add 100 microliters of the stop solution to stop the digestion reaction and acidify the peptide solution. Using a tube adapter, place a desalting column in a 1.7-milliliter low protein binding micro centrifuge tube.Add the entire acidified peptide solution to the column. Spin the column at 3, 800 G for 3 minutes, so that the solution flows through the column. Discard the flow-through as the peptides are now bound to the column.Add 200 microliters of Wash 1 solution to the column, spin at 3, 800 G for 3 minutes at room temperature, and then discard the flow-through. Repeat the washing step with 200 microliters of Wash 2 solution. Transfer the column to a new labeled 1.7-milliliter low protein binding micro centrifuge tube.Add 100 microliters of elute solution to the column and spin at 3, 800 G for 3 minutes at room temperature. The peptides are now eluted off the column into the tube. Place the peptide solution in a vacuum centrifuge and allow it to dry overnight.Place the dried down peptides at minus 80 degrees Celsius until ready to quantify and analyze on the mass spectrometer. Proteome analysis was performed to characterize the enriched cell surface proteins using liquid chromatography tandem mass spectrometry. A final peptide concentration of approximately 630 nanograms per microliter was obtained based on the colorimetric peptide quantification assay.Mass spectrometry data analyzed using MaxQuant and subsequent dataset analysis of the cell surface proteins yielded 601 surface proteins in the sample, approximately 27%of all the proteins identified. A plot representing the distribution of the identified protein in Andromeda score is shown here, and the scatter plot illustrates the sample-to-sample correlation. The box plots depicted the run-to-run variation.And the sample-wise distribution plot represented the data quality of replicates. The most important thing to keep in mind is where the peptides are at each step. Initially, they're in solution, then, they're bound to the beads until the digestion, then, they're bound to desalting column until they're finally eluted.Following this procedure, there are various ways to validate a handful of the proteins identified, such as targeted flow cytometry and targeted proteomics. This will give more detailed information about the expression levels of these proteins across the sample.

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Research

JoVE Journal

Biochemistry

1.8K visualizações.  University of California, San Francisco. This protocol allows us to investigate antigens present on the cell surface of various cell types to find antigens specific to that type of cell or tissue. The technique we present here allows for enrichment of cell membrane proteins out of a whole cell lysate, ultimately for analysis by mass spectrometry-based proteomics. So one specific application of this protocol is to apply it to tumor cells to look for antigens present on the surface of those cancer cells and not normal cells that could...