This research has been supported by the startup fund of Simon Fraser University. &#160;

1. Harvest Membranes
<ol>
	<li>Add hypotonic buffer (10 mM Tris-HCl, 10 mM KCl, 1.5 mM MgCl2, pH 8.0) with protease inhibitor cocktail onto a cell pellet (~108 cells) and incubate for 15-30 min on ice. Lyse the cells by passing the sample through syringe needles (5-10 passes) or homogenizing the sample by a Dounce homogenizer (15-30 strokes). Use a hemocytometer and trypan blue staining to check the efficiency of lysis.</li>
	<li>Obtain the microsomal fraction by a differential centrifugation of t...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation,+immunity,+and+autoimmunity.">Glycosylation, immunity, and autoimmunity.</a> Cell. 104, 809-812 (2001).</li><li>Dodds, E. 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The authors declare that they have no competing financial interests. &#160;

Here we introduce a glycopeptide-capture strategy for profiling cell-surface proteins. The method can be applied to study secreted proteins, such as those in blood, as well as in other body fluids or in cell culture media.
The success of the method relies on the complete digestion of samples; therefore, a SDS-PAGE characterization of the digestion efficiency is necessary, especially for the first-time analysis of a sample. A complete digestion can be challenging for membrane proteins, and can ...

Cell surface proteins interact with the extracellular environment and relay signals from the outside to the inside of a cell. Thus, these proteins,&#160;including extracellular matrix proteins,&#160;play critical roles in all aspects of cellular biology and physiology ranging from proliferation, growth, migration, differentiation to aging and so forth. Surface proteins function by interacting with other cells, proteins and small molecules1-3. Molecular characterization of cell-surface proteins is of great interest not only for biologists but also for pharmaceutical companies, as more than 60% of drugs are targeted to cell-surface proteins4.
Tandem mass spectrometry (MS), with its superior sensitivity, accuracy, and throughput for identification of proteins and peptides, has been a powerful tool for global proteomic studies5,6. Yet, surface proteins pose significant challenges to MS-based proteomics, as most surface proteins exist in low quantities and with heavy modifications. The membrane-spanning regions of the surface proteins render them hydrophobic; this is especially the case for multipass transmembrane proteins. It is thus difficult to dissolve membrane proteins in aqueous solutions without the help of a detergent; however the use of detergents generally suppresses the performance of HPLC and MS1,7,8 in protein identification. Therefore, membrane proteins have been poorly characterized in direct LC-MS based proteomics.
Glycosylation is one of the most important and abundant post-translational modifications taking place in cell-surface proteins9. The enormous complexity and heterogeneity of glycans hamper peptides&#8217; MS signal10. Nevertheless, several proteomic methods have used this unique modification to enrich surface proteins and to remove the sugar moieties from proteins prior to LC-MS analysis. These methods include lectin-based affinity capture11 and hydrazide-based or boric acid-based chemical capture12 as well as hydrophilic chromatography separations8,13. The removal of glycans transforms membrane proteins to regular proteins and drastically simplifies the MS characterization. Because glycosylation also takes place in secreted proteins that have high solubility in contrast to membrane proteins, many glycoproteomic methods are optimized for soluble proteins, and tend to have lower glycopeptide selectivity and sensitivity when being deployed to membrane proteins8,14. Other methods also exist to enrich, in particular, cell-surface proteins, such as those using ultracentrifugation15 and labeling strategies16. A detailed comparison between our method and other existing methods for characterizing membrane proteins was conducted recently17, and the results indicated that our method can perform equally well, if not better, than all the compared membrane proteomics methods, but with higher simplicity.
To help researchers use this method, we detail here a general protocol. This method integrates several advantages of existing glycoproteomics strategies and is devised specifically for membrane glycoproteins, yet the method works equally well for secreted proteins. The characteristics of this method include: 1) a complete solubilization of membrane proteins, 2) an enrichment of glycopeptides instead of glycoproteins to eliminate the potential steric hindrance when using a solid capturing substrate, 3) the use of hydrazide chemistry to form covalent bonds between glycopeptides and the capturing substrate, such that the bonded glycopeptides can tolerate stringent washes for high glycoselectivity, and 4) the capability to conduct the entire capture procedure in one tube for reduced sample loss and shortened procedure duration. After implementing this method to studying a variety of biological samples including cells and tissues, we observed a high selectivity (&#62; 90%) to glycoproteins8,17,18.

<table border="0" cellpadding="0" cellspacing="0" width="701">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DTT</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iodoacetamide</td>
			<td style="width:199px;">Sigma</td>
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			<td height="21" style="height:21px;width:216px;">Sodium periodate</td>
			<td style="width:199px;">Sigma</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PNGase F</td>
			<td style="width:199px;">New England Biolabs</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Affi-Gel Hz Hydrazide gel</td>
			<td style="width:199px;">Bio-Rad</td>
			<td style="width:119px;">153-6047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:199px;">Worthington Biomedical</td>
			<td style="width:119px;">LS02115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sep-Pak C-18 cartridge</td>
			<td style="width:199px;">Waters</td>
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			<td height="21" style="height:21px;width:216px;">Oasis MCX cartridge</td>
			<td style="width:199px;">Waters</td>
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			<td height="21" style="height:21px;width:216px;">Protease inhibitor coctail</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:199px;">Amersco</td>
			<td style="width:119px;">568</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium sulphite</td>
			<td style="width:199px;">Caledon</td>
			<td style="width:119px;">8360-1</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Invertase</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">I0408</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alpha-1 trypsin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ribonulease B</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">R7884</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Avidin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A9275</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ovalbumin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">A5503</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conalbumin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:119px;">C0755</td>
			<td></td>
		</tr>
	</tbody>
</table>

glycopeptide capture for cell surface proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

A representative flow chart of the experimental procedure is summarized in Figure 1. The labeling and further fractionation steps are optional and details are described in a recent publication18. Another option is to analyze the unmodified peptides, which do not react with the resin. The advantages of analyzing the unmodified peptides include the potential identification of non-glycosylated peptides and proteins, such as claudins in tight junctions; an additional advantage is more accurate qua...

Watch this Scientific Journal Video about Glycopeptide Capture for Cell Surface Proteomics at JoVE.com

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

glycopeptide-capture-for-cell-surface-proteomics

Research

JoVE Journal

Biology

11.2K visualizações.  Simon Fraser University.  As proteínas de superfície são biologicamente importantes e amplamente glicosilada. Apresentamos aqui uma abordagem de captura de glycopeptide para solubilizar, enriquecer e deglycosylate estas proteínas para análise proteômica baseada LC-MS fáceis. 