Name: Glycopeptide Capture for Cell Surface Proteomics (Video) | JoVE Journal
Uploaded: 2014-05-09T20:00:00.000+00:00
Duration: 10 min 11 s
Description: Learn glycopeptide capture for cell surface proteomics using membrane protein digestion and LC-MS analysis. This method enables enrichment and quantification of low abundance glycosylated proteins.

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5 minutes

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 the lysate at 3,000 g x 15 min and then at 100,000 g x 2 hr. The first centrifugation obtains a pellet that is the nuclear fraction, and the subsequent centrifugation of the supernatant generates a second pellet, which is the microsomal fraction that contains the plasma membrane as well as membrane-bound organelles8. The final supernatant is the cytosolic fraction. Store the microsomal fractions in -80 &#176;C freezer for further analysis as indicated below.</li>
</ol>
2. Dissolve, Denature, and Digest Membrane Proteins
<ol>
	<li>Dissolve the microsomal fraction in the denaturation buffer (40 mM Tris, 10 mM EDTA, 10 mM TCEP, 0.5% Rapigest, pH 8) and then incubate the solution at 100 &#176;C for 10 min.</li>
	<li>Cool the heated solution to room temperature, add ultra-high purity urea powder into the solution to 8 M final concentration and incubate the sample at 37 &#176;C for 30 min.</li>
	<li>Add iodoacetamide stock solution to the sample to 15 mM final concentration, and incubate the solution in the dark for 30 min at room temperature to alkylate the free thiols in the sample. Add DTT stock solution to the sample to 10 mM final concentration and incubate the solution for another 10 min at room temperature to quench the excessive iodoacetamide.</li>
	<li>Dilute the obtained solution 10x&#160;with 40 mM Tris buffer at pH 8, and subsequently add trypsin to the sample at a 1:20 ratio of trypsin to total protein. Maintain the digestion reaction in a 37 &#176;C oven overnight to ensure the reaction is completed.</li>
	<li>Terminate the digestion by acidifying the sample solution to pH 1 with HCl, a condition that also breaks down the detergent, i.e. Rapigest. Degrade the Rapigest at 37 &#176;C for 1 hr, and remove the developed precipitation by centrifugation.</li>
	<li>Clean the supernatant that contains sample peptides by a C-18 solid phase extraction (SPE) cartridge and dry the obtained sample by speedvac.</li>
	<li>Perform a SDS-PAGE analysis of samples before and after trypsin digestion to confirm the digestion efficiency.</li>
</ol>
3. Glycopeptide Capture
<ol>
	<li>Dissolve the cleaned peptides in the coupling buffer (100 mM sodium acetate, pH 5.5). Add sodium periodate into the peptide solution at a 10 mM final concentration for 30 min in the dark, at room temperature. This will oxidize the cis-diol groups in the glycans to aldehydes, which allow the glycans to couple to the resin through hydrazide chemistry. Quench the excessive periodate by sodium sulphite at a 20 mM final concentration and pH 5 for 10 min at room temperature.</li>
	<li>Introduce hydrazide-derivatized resin into the peptide solution at a ratio of 1:4 (resin to solution) to couple glycopeptides to the resin. Incubate the reaction at 37 &#176;C for 1-2 days with end-to-end rotation for complete coupling.</li>
	<li>Remove the unbound peptides by washing the resin twice with 1 ml of each of the following: DI&#160;water, 1.5 M NaCl, methanol and 80% acetonitrile. Finally, wash the resin 3x&#160;with 1 ml of 100 mM NH4HCO3 at pH 8 to exchange the buffer of the system to 100 mM NH4HCO3.
	<ol>
		<li>Collect the supernatant and the washes for the analysis of unbound peptides (optional).</li>
	</ol>
	</li>
	<li>Release the N-glycopeptides from the resin by PNGase F in an overnight incubation at 37 &#176;C with an end-to-end rotation. Collect the released peptides by centrifugation and an 80% acetonitrile wash. Dry the obtained solution in the speedvac for LC-MS analysis.</li>
</ol>
4. Further Fractionation (Optional)
To further simplify sample complexity, fractionate the obtained N-glycopeptides. For example, redissolve the dried peptides into 10 mM ammonia formate, pH 3 with 20% acetonitrile and use strong&#160;cation&#160;exchange (SCX) chromatography to fractionate the peptides. Dry the obtained eluent, and then analyze the obtained peptide fractions by reverse-phase LC-MS8,17,18.
5. Cleaning of the Released N-glycopeptides (Optional)
If concerns rise for the potential contamination of the peptides, redissolve the dried peptides into 0.1% formic acid and use a MCX SPE column to further clean the peptides prior to reverse-phase LC-MS analysis.
Note: Database searching parameters.
During the selective cleavage of N-glycopeptides off the resin, PNGase F converts the N-glycan linked asparagine to an aspartic acid. Therefore, there is a 0.9840 Da mass shift of the liberated N-glycopeptides. To accurately identify these peptides, this modification needs to be added to the search parameters along with common modifications such as the carbamidomethylation of the cysteine and oxidation of the methionine.

<ol><li>Cho, W., Stahelin, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane-protein+interactions+in+cell+signaling+and+membrane+trafficking%5BTitle%5D)+AND+%22Annual+review+of+biophysics+and+biomolecular+structure%22%5BJournal%5D)">Membrane-protein interactions in cell signaling and membrane trafficking.</a> Annual review of biophysics and biomolecular structure. 34, 119-151 (2005).</li>
<li>Tadevosyan, A., Vaniotis, G., Allen, B. G., Hebert, T. E., Nattel, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((protein-coupled+receptor+signalling+in+the+cardiac+nuclear+membrane%3A+evidence+and+possible+roles+in+physiological+and+pathophysiological+function%5BTitle%5D)+AND+%22The+Journal+of+physiology%22%5BJournal%5D)">protein-coupled receptor signalling in the cardiac nuclear membrane: evidence and possible roles in physiological and pathophysiological function.</a> The Journal of physiology. 590, 1313-1330 (2012).</li>
<li>White, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biophysical+dissection+of+membrane+proteins%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Biophysical dissection of membrane proteins.</a> Nature. 459, 344-346 (2009).</li>
<li>Heijne, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane-protein+topology.+%5BTitle%5D)+AND+%22Nature+reviews+Molecular+cell+biology%22%5BJournal%5D)">Membrane-protein topology. </a> Nature reviews Molecular cell biology. 7, 909-918 (2006).</li>
<li>Cox, J., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+high-resolution+proteomics+for+data-driven+systems+biology%5BTitle%5D)+AND+%22Annual+review+of+biochemistry%22%5BJournal%5D)">Quantitative high-resolution proteomics for data-driven systems biology.</a> Annual review of biochemistry. 80, 273-299 (2011).</li>
<li>Lamond, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advancing+cell+biology+through+proteomics+in+space+and+time+%28PROSPECTS%29%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Advancing cell biology through proteomics in space and time (PROSPECTS).</a> Mol Cell Proteomics. 11, (2012).</li>
<li>Savas, J. N., Stein, B. D., Wu, C. C., Yates 3rd, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometry+accelerates+membrane+protein+analysis%5BTitle%5D)+AND+%22Trends+in+biochemical+sciences%22%5BJournal%5D)">Mass spectrometry accelerates membrane protein analysis.</a> Trends in biochemical sciences. 36, 388-396 (2011).</li>
<li>Sun, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shotgun+glycopeptide+capture+approach+coupled+with+mass+spectrometry+for+comprehensive+glycoproteomics%5BTitle%5D)+AND+%22Mol+Cell+Proteomics%22%5BJournal%5D)">Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics.</a> Mol Cell Proteomics. 6, 141-149 (2007).</li>
<li>Lowe, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycosylation%2C+immunity%2C+and+autoimmunity%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Glycosylation, immunity, and autoimmunity.</a> Cell. 104, 809-812 (2001).</li>
<li>Dodds, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gas-phase+dissociation+of+glycosylated+peptide+ions%5BTitle%5D)+AND+%22Mass+spectrometry+reviews%22%5BJournal%5D)">Gas-phase dissociation of glycosylated peptide ions.</a> Mass spectrometry reviews. 31, 666-682 (2012).</li>
<li>Kaji, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lectin+affinity+capture%2C+isotope-coded+tagging+and+mass+spectrometry+to+identify+N-linked+glycoproteins%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins.</a> Nat Biotechnol. 21, 667-672 (2003).</li>
<li>Zhang, H., Li, X. J., Martin, D. B., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+quantification+of+N-linked+glycoproteins+using+hydrazide+chemistry%2C+stable+isotope+labeling+and+mass+spectrometry%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.</a> Nat Biotechnol. 21, 660-666 (2003).</li>
<li>Hagglund, P., Bunkenborg, J., Elortza, F., Jensen, O. N., Roepstorff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+strategy+for+identification+of+N-glycosylated+proteins+and+unambiguous+assignment+of+their+glycosylation+sites+using+HILIC+enrichment+and+partial+deglycosylation%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation.</a> J Proteome Res. 3, 556-566 (2004).</li>
<li>Bond, M. R., Kohler, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+methods+for+glycoprotein+discovery%5BTitle%5D)+AND+%22Current+opinion+in+chemical+biology%22%5BJournal%5D)">Chemical methods for glycoprotein discovery.</a> Current opinion in chemical biology. 11, 52-58 (2007).</li>
<li>Pasini, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In-depth+analysis+of+the+membrane+and+cytosolic+proteome+of+red+blood+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">In-depth analysis of the membrane and cytosolic proteome of red blood cells.</a> Blood. 108, 791-801 (2006).</li>
<li>Wollscheid, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass-spectrometric+identification+and+relative+quantification+of+N-linked+cell+surface+glycoproteins%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins.</a> Nat Biotechnol. 27, 378-386 (2009).</li>
<li>Sun, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((N-Glycoproteome+of+E14.Tg2a+mouse+embryonic+stem+cells%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">N-Glycoproteome of E14.Tg2a mouse embryonic stem cells.</a> PLoS ONE. 8, (2013).</li>
<li>Sun, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycocapture-assisted+global+quantitative+proteomics+%28gagQP%29+reveals+multiorgan+responses+in+serum+toxicoproteome%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">Glycocapture-assisted global quantitative proteomics (gagQP) reveals multiorgan responses in serum toxicoproteome.</a> J Proteome Res. 12, 2034-2044 (2013).</li>
</ol>

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><tbody><tr><td>DTT</td><td>Sigma</td><td>646563</td><td></td></tr><tr><td>TCEP</td><td>Sigma</td><td>646547</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma</td><td>A3221</td><td></td></tr><tr><td>Rapigest SF</td><td>Waters</td><td>186001860</td><td></td></tr><tr><td>Sodium periodate</td><td>Sigma</td><td>311448</td><td></td></tr><tr><td>PNGase F</td><td>New England Biolabs</td><td>P0704S</td><td></td></tr><tr><td>Affi-Gel Hz hydrazide gel</td><td>Bio-Rad</td><td>153-6047</td><td></td></tr><tr><td>Trypsin</td><td>Worthington Biomedical</td><td>LS02115</td><td></td></tr><tr><td>Sep-Pak C-18 cartridge</td><td>Waters</td><td>WAT054955</td><td></td></tr><tr><td>Oasis MCX cartridge</td><td>Waters</td><td>186000252</td><td></td></tr><tr><td>Protease inhibitor coctail</td><td>Sigma</td><td>P8340</td><td></td></tr><tr><td>Urea</td><td>Amersco</td><td>568</td><td></td></tr><tr><td>Sodium sulphite</td><td>Caledon</td><td>8360-1</td><td></td></tr><tr><td>Invertase</td><td>Sigma</td><td>I0408</td><td></td></tr><tr><td>&amp;alpha;-1 trypsin</td><td>Sigma</td><td>F2006</td><td></td></tr><tr><td>Ribonulease B</td><td>Sigma</td><td>R7884</td><td></td></tr><tr><td>Avidin</td><td>Sigma</td><td>A9275</td><td></td></tr><tr><td>Ovalbumin</td><td>Sigma</td><td>A5503</td><td></td></tr><tr><td>Conalbumin</td><td>Sigma</td><td>C0755</td><td></td></tr></tbody></table>

glycopeptide capture for cell surface proteomics

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...

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, ...

The overall goal of this procedure is to enrich low abundance cell surface membrane proteins. This is accomplished by first denaturing and digesting membrane proteins. The second step is to oxidize the glycans and capture the glycosylated peptides on a resin surface.The non glycopeptides are then washed off the resin. The final step is release of the N-Linked Glycopeptides off the resin for lc MS analysis. Ultimately, the identified peptide sequence can be mapped back to proteins for identification and quantification purposes.Today I'm going to demonstrate you how the Geico peptide capture ral works. The main advantage of this technique overdose, ex existent ones, such as glycoprotein capture, is that the membrane protein will first be digested and the capture of the glycopeptide can be performed in one vessel. The application of this technique can extend to the discovery of the disease biomarker.Since gator protein can be shed in the blood and other body fluid and the circulating glycoprotein in blood, which are specific to the pathological process, can be used as prognosis and diagnosis Biomarkers. First, prepare four milliliters of hypotonic buffer with one to 100 protease inhibitor cocktail. Add one milliliter of hypotonic buffer onto a cell pellet containing approximately 10 to the eighth cells, and incubate the mixture for 15 to 30 minutes On ice, transfer the cells into a two milliliter vial and lice the cells by passing the sample five to 10 times through a syringe needle.Then use a hemo cytometer and trian blue staining. To check the efficiency of lysis, combine the one milliliter lysate with three milliliters of leftover hypotonic buffer. Spin the tube at 3000 times G at four degrees Celsius for 15 minutes.Transfer the super supernatant to an ultracentrifuge tube and store the pellet in a negative 80 degrees Celsius freezer. Spin the ultracentrifuge tube at 100, 000 times G at four degrees Celsius for two hours. Next, dissolve the microsomal fraction in 200 microliters of denaturation buffer, and then boil the solution at 100 degrees Celsius for 10 minutes.After cooling the heated solution to room temperature, add ultra high purity urea powder to give an eight molar final concentration and incubate the sample at 37 degrees Celsius for 30 minutes. After incubation, add 500 millimolar. I oto Acetamide stock solution to the sample to give a final concentration of 15 millimolar.Then incubate the solution in the dark for 30 minutes at room temperature to alkylate the free thiols in the sample. Following this, add one molar DTT stock solution to the sample to give a final concentration of 10 millimolar and incubate the solution for another 10 minutes at room temperature. To quench the excessive I oto Acetamide dilute the obtained solution 10 times with 40 millimolar tris buffer at PH eight, and subsequently add trypsin to the sample at a one to 20 ratio of trypsin to total protein quantity.Obtain the quantity of total protein using the Bradford assay. Maintain the digestion reaction in a 37 degrees Celsius oven overnight to ensure the reaction is completed. When finished, terminate the digestion by acidifying the sample solution to pH one with 10 millimolar hydrochloric acid, a condition that also degrades the rapidest.The detergent then degrade the rapidest at 37 degrees Celsius for one hour in an incubator. After incubation, remove the developed precipitation by centrifugation following centrifugation. Clean the SUP natin that contains the sample peptides by a C 18 solid phase extraction cartridge and dry the obtained sample by speed vac.Perform an SDS page analysis of samples before and after trips and digestion to confirm the digestion efficiency. At this point, dissolve the cleaned peptides in 200 microliters of coupling buffer. After adding sodium puriate to the peptide solution to give a 10 millimolar final concentration.Incubate the sample for 30 minutes in the dark at room temperature When the incubation is complete, quench the excessive prate with sodium sulfite to give a final concentration of 20 millimolar and pH of five following incubation for 10 minutes at room temperature, introduce 200 microliters of hydrocyte derivative resin solution, pree equilibrated and coupling buffer into the peptide solution. Incubate the reaction at 37 degrees Celsius for one to two days with end-to-end rotation for complete coupling. When finished, remove the unbound peptides by washing the resin twice with one milliliter of deionized water, one milliliter of 1.5 molar sodium chloride, one milliliter of methanol, and one milliliter of 80%aceto nitrile.Next, collect the supernatant and the washes for the analysis of unbound peptides. Store the samples in a minus 80 degrees Celsius freezer until analysis. Then wash the resin three times with one milliliter of 100 millimolar ammonium bicarbonate at pH eight.Finally, add 300 microliters of ammonium bicarbonate into the sample vial. To release the N glycopeptides from the resin. Add PNG ACE F and incubate the sample at 37 degrees Celsius with an end-to-end rotation.Following this, collect the released peptides by centrifugation, then wash the resin with one milliliter of 80%acetyl nitrile. Combine the wash with the previously collected. Finally dry the obtained solution in the speed VAC for lc MS analysis.Atypical glycopeptide spectrum taken after the enrichment method is shown here in the obtained glycopeptide. The n glycan was removed and the glycan attached disparaging was converted to an spartic acid by P-N-G-A-F. Therefore, the spectrum can be readily searched by any proteomic search engine against common protein sequence databases.Atypical lc MS.Result of the captured n glycopeptides is shown here in which more than 100 glycoproteins can be identified from a single lc MS run of a cell microsomal fraction. The enrichment selectivity to both glycoproteins and glycopeptides is generally more than 90%A successful analysis can have an enrichment selectivity of 95%using an SCX column and step gradient to further fractionate. The samples prior to LCMS analysis will usually double the number of glycoprotein identifications.Sometimes when the quantity of the obtained glycopeptides is low, the impurity accumulated from the vials can be observed in the final sample. These contaminants can be removed by an MCX column. So after watching this video, I hope you have a good understanding of how to use our glycopeptide capture methods to study self-service membrane protein.While you're attempting this protocol, it is also important to set aside samples for each step for troubleshooting. The volume of the reagent can also be optimized for your specific study.

glycopeptide-capture-for-cell-surface-proteomics

Research

JoVE Journal

Biology

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

A Quantitative Glycomics and Proteomics Combined Purification Strategy

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model.

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic tissues.

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

A protocol is presented for the determination of low-abundance biomarkers from dried serum samples exemplified with the biomarker progastrin-releasing peptide (ProGRP). Antibody-coated magnetic beads are used for the selective cleanup and enrichment of a proteotypic ProGRP peptide. The captured peptide is subsequently analyzed by liquid chromatography-tandem mass spectrometry.

Microsampling in Targeted Mass Spectrometry-Based Protein Analysis of Low-Abundance Proteins

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

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Learn glycopeptide capture for cell surface proteomics using membrane protein digestion and LC-MS analysis. This method enables enrichment and quantification of low abundance glycosylated proteins.

Glycopeptide Capture for Cell Surface Proteomics

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