Glycopeptide Capture for Cell Surface Proteomics

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

Cell surface proteins interact with the extracellular environment and relay signals from the outside to the inside of a cell. Thus, these proteins, including extracellular matrix proteins, 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 molecules^1-3. 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 studies^5,6. 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 MS^1,7,8 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’ 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 separations^8,13. 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 proteins^8,14. 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 (> 90%) to glycoproteins^8,17,18.

Protokół

1. Harvest Membranes

1. 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.
2. 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 °C freezer for further analysis as indicated below.

2. Dissolve, Denature, and Digest Membrane Proteins

1. 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 °C for 10 min.
2. 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 °C for 30 min.
3. 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.
4. Dilute the obtained solution 10x 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 °C oven overnight to ensure the reaction is completed.
5. 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 °C for 1 hr, and remove the developed precipitation by centrifugation.
6. Clean the supernatant that contains sample peptides by a C-18 solid phase extraction (SPE) cartridge and dry the obtained sample by speedvac.
7. Perform a SDS-PAGE analysis of samples before and after trypsin digestion to confirm the digestion efficiency.

3. Glycopeptide Capture

1. 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.
2. 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 °C for 1-2 days with end-to-end rotation for complete coupling.
3. Remove the unbound peptides by washing the resin twice with 1 ml of each of the following: DI water, 1.5 M NaCl, methanol and 80% acetonitrile. Finally, wash the resin 3x with 1 ml of 100 mM NH₄HCO₃ at pH 8 to exchange the buffer of the system to 100 mM NH₄HCO₃.
   1. Collect the supernatant and the washes for the analysis of unbound peptides (optional).
4. Release the N-glycopeptides from the resin by PNGase F in an overnight incubation at 37 °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.

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 cation exchange (SCX) chromatography to fractionate the peptides. Dry the obtained eluent, and then analyze the obtained peptide fractions by reverse-phase LC-MS^8,17,18.

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.

Wyniki

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...

Dyskusje

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 ...

Materiały

Name	Company	Catalog Number	Comments
DTT	Sigma	646563
TCEP	Sigma	646547
Iodoacetamide	Sigma	A3221
Rapigest SF	Waters	186001860
Sodium periodate	Sigma	311448
PNGase F	New England Biolabs	P0704S
Affi-Gel Hz hydrazide gel	Bio-Rad	153-6047
Trypsin	Worthington Biomedical	LS02115
Sep-Pak C-18 cartridge	Waters	WAT054955
Oasis MCX cartridge	Waters	186000252
Protease inhibitor coctail	Sigma	P8340
Urea	Amersco	568
Sodium sulphite	Caledon	8360-1
Invertase	Sigma	I0408
α-1 trypsin	Sigma	F2006
Ribonulease B	Sigma	R7884
Avidin	Sigma	A9275
Ovalbumin	Sigma	A5503
Conalbumin	Sigma	C0755