Chapter 43 N-Glycoprotein Enrichment by Lectin Affinity Chromatography Eliel Ruiz-May, Carmen Catalá, and Jocelyn K.C. Rose Abstract Lectins are proteins that bind to sugars with varying specificities and several have been identified that show differential binding to structurally variable glycans attached to glycoproteins. Consequently, lectin affinity chromatography represents a valuable tool for glycoproteome studies, allowing enrichment of glycoproteins in samples prior to their identification by mass spectrometry (MS). From the perspective of plant scientists, lectin enrichment has proven useful for studies of the proteomes of the secretory pathways and cell wall, due to the high proportion of constituent proteins that are glycosylated. This chapter outlines a strategy to generate samples enriched with glycoproteins from bulk plant tissues prior to further characterization by MS, or other techniques. Key words Glycoprotein, Lectins, Affinity chromatography, Concanavalin A
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Introduction Glycosylation is a highly complex posttranslational modification associated with many eukaryotic proteins, involving the attachment of oligosaccharide moieties and their subsequent modification by a large battery of glycan modifying enzymes. This results in structurally diverse pool of glycoproteins and glycoforms [1, 2]. In plants, these glycoproteins can be classified in N-glycoproteins, where N-glycans are covalently linked to asparagine in the sequon N-X-(S/T), where X can be any amino acid except proline [3], and O-glycoproteins, where in the glycan is attached to the hydroxyl group of serine, threonine, or hydroxylated proline residues [4–7], with no specific sequon. Several analytical platforms have been developed for systematic studies of glycoproteins from bacteria, yeast and animals [8–14], but there are not yet an analogous system for plant glycoproteomes. A typical workflow might comprise front-end enrichment of glycoproteins/glycopeptides, identification of peptide sequence, determination of glycosylation sites and
Jesus V. Jorrin-Novo et al. (eds.), Plant Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 1072, DOI 10.1007/978-1-62703-631-3_43, © Springer Science+Business Media, LLC 2014
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Fig. 1 Strategy workflow for systematic study of N-glycoproteins
site occupancy, and interpretation of glycan structure and glycoforms (Fig. 1). However, several factors can complicate the glycoprotein analysis, including the complexity of biological samples and low protein abundance. Therefore, the reduction of the complexity and enrichment of glycoproteins can represent an effective first step. This can be accomplished by lectin affinity chromatography [15–17] and chemical methods, such as hydrazine chemistry [18] and boronic acid [19]. However, lectin affinity chromatography with Concanavalin A has been more commonly used for studies of plant glycoproteins [15–17] and a number of glycoproteomic analyses have been reported in the last few years employing lectin affinity as an early enrichment step [20], including those that use multiple lectins to increase the population of captured glycoproteins [17]. To this end, a range of lectins with different affinities is now commercially available (Table 1). However, the chemical methods should be considered as complementary analytical approaches for a systematic study [14, 21], although they are not further discussed here. In this chapter we present a protocol to enrich for N-glycoproteins from plant tissues using lectin affinity chromatography, and to prepare the samples for downstream experiments designed to identify the proteins using mass spectrometry (MS).
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Table 1 Commercially available glycan-binding lectins for the enrichment of glycoproteins Lectin type
Name
Source
Affinity
Mannose binding lectins
Con A (concanavalin A)
Canavalia ensiformis
High-mannose, hybrid and biantennary complex type N-glycans [30–33]
LCH (Lentil lectin)
Lens culinaris
Fucosylated core region of bi- and triantennary complex type N-glycans [34]
GNA (Snowdrop lectin)
Galanthus nivalis
α-1,3 and α-1,6 link high mannose structure [35]
UEA (Ulex europaeus agglutinin)
Ulex europaeus
Fucα1-2Gal-R [36]
AAL (Aleuria aurantia)
Aleuria aurantia
Fucα1-2Galβ1-4(Fucα13/4) Galβ1-4GlcNAc; R2GlcNAcβ1-4(Fucα1 6) GlcNAc-R1 [37]
RCA (Ricinus communis Agglutinin)
Ricinus communis
Galβ1-4GlcNAcβ1-R [38, 39]
PNA (Peanut Agglutinin)
Arachis hypogaea
Galβ1-3GalNAcα1-Ser/Thr (T-Antigen) [40–42]
AIL (Jacalin)
Artocarpus integrifolia
(Sia)Galβ1-3GalNAcα1-Ser/ Thr (T-Antigen) [43]
VVL (Hairy vetch lectin)
Vicia villosa
GalNAcα-Ser/Thr (Tn-Antigen) [44, 45]
WGA (Wheat Germ agglutinin)
Triticum vulgaris
GlcNAcβ1-4GlcNAcβ14GlcNAc, Neu5Ac (sialic acid) [46, 47]
SNA (Elderberry lectin)
Sambucus nigra
Neu5Acα2-6Gal(NAc)-R [48]
MAL (Maackia amurensis lectin)
Maackia amurensis
Neu5Ac/Gcα2-3Galβ14GlcNAcβ1-R [49]
Fucose binding lectins
Galactose/Nacetylgalactosamine binding lectins
Sialic acid/Nacetylglucosamine binding lectins
2
Materials Note: mention of specific companies or pieces of equipment does not represent an endorsement by the authors.
2.1
Lectin Resins
Several lectins coupled to Sepharose beads, magnetic beads [22, 23] or to beads packed into chromatography columns/cartridges are commercially available (e.g., Qiagen) to facilitate the enrichment of a diverse population of glycoproteins (Table 1). Of these, Concanavalin A (Con A) is the most frequently used. It is possible to work with several lectins sequentially [24, 25], in parallel [8, 14, 26]
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Fig. 2 Different modalities of lectin affinity chromatography. Lectin enrichment can be done in parallel (a), sequentially (b), and using a mix of several lectins (c). The type of lectins used can be tailored to the kind of glycoprotein that is being targeted
and as mixtures [27], as illustrated in Fig. 2. If the main goal is to enrich for N-glycoproteins an effective option is the use of mannose binding lectins (Table 1). 2.2 Porous Graphitic Carbon (PGC) Columns
Prepacked PGC columns (1 mL, brand name Hypersep Hypercarb) can be purchased from Thermo Scientific.
2.3
Buffer solutions must be prepared with bi-distilled water, filtered with 0.45 μm filters, degassed, and precooled to 4 °C before starting the main protocol.
Buffer Solutions
1. Stock buffers: prepare 1 M Tris, pH 7.0 (Solution A) and 5 M NaCl (Solution B) solutions. Dissolve 121.4 g Tris in 900 mL water, and adjust the pH with HCl, and the final volume to 1 L with water. Dissolve 292 g NaCl in 500 mL water and adjust the volume to 1 L. 2. Protein Extraction buffer: 25 mM Tris, pH 7.0, 0.5 M NaCl, 0.2 M CaCl2, and 20 μL/g fresh weight protease inhibitor cocktails. For 100 mL of protein extraction buffer mix: 2.5 mL A solution, 10 mL B solution, 2.94 g CaCl2, and 20 μL/g fresh weight protease inhibitor cocktails and adjust the volume to 100 mL (see Note 1). 3. Binding buffer (Lectin): 20 mM Tris–HCl, 0.5 M NaCl, 1 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2. Mix 2 mL Solution
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A, 10 mL Solution B, 0.0147 g CaCl2, 0.0197 g MnCl2, and 0.0203 g MgCl2 and adjust the volume to 100 mL. 4. Elution buffer (Lectin): binding buffer plus 0.5 M α-methyl-Dmannopyranoside. Dissolve 9.71 g α-methyl-D-mannopyranoside in 100 mL binding buffer. 5. 100 mM Ammonium bicarbonate (Na2CO3): dissolve 1.05 g Na2CO3 in water and adjust the final volume to 100 mL. 6. Resuspension buffer: 8 M urea, 100 mM Na2CO3. Dissolve 0.961 g urea in 2 mL 100 mM Na2CO3. Prepare this solution fresh immediately prior to use. 7. 2 M Dithiothreitol (DTT): dissolve 0.154 g DTT in 100 mM Na2CO3, adjust the final volume to 500 μL, divide into small aliquots (e.g., 5 μL) and store at −80 °C until use. 8. 257 mM Iodoacetamide: dissolve 0.023 g iodoacetamide in 100 mM Na2CO3 and adjust the final volume 500 μL. Prepare this solution fresh immediately prior to use. 9. 1 M NaOH: dissolve 4 g NaOH in water and adjust the volume to 100 mL. 10. 30 % Acetic acid: mix 30 mL glacial acetic acid with 70 mL water. This solution should be prepared in a fume hood to avoid potentially toxic fumes. 11. Porous graphitic Carbon (PGC) wash solvent: 5 % acetonitrile, 0.1 % formic acid (v/v) in water. Mix 5 mL acetonitrile and 100 μL formic acid and adjust the volume to 100 mL with water. This solution should be prepared in the fume hood. 12. PGC Elution solvent: 50 % acetonitrile, 0.1 % formic acid (v/v) in water. Mix 50 mL and 100 μL formic acid and adjust the volume to 100 mL with water. This solution should be prepared in the fume hood.
3 3.1
Methods Tissue Collection
3.2 Protein Extraction
1. Weigh out 3 g of plant material (fresh or flash frozen in liquid nitrogen and stored at −80 °C). Replicated biological samples should also be considered, depending on the experimental goals. 1. Powder the samples in liquid nitrogen using a pestle and mortar and add a tenth mass of polyvinylpolypyrrolidone (PVPP; 1 g/10 g fresh weight) to help remove phenolic compounds. 2. Homogenize the material in three volumes of extraction buffer (45 mL) with a tissue homogenizer (e.g., Polytron, Kinematica) for 15 s, and then filter with Miracloth (Calbiochem). Shake the crude extract at 5 rpm on a rocking platform for 2 h at 4 °C.
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Fig. 3 A typical UV absorbance chromatogram of the N-glycoprotein enriched fraction from a crude protein extract
3. Centrifuge the crude extract at 15,000 × g for 30 min. Recover the supernatant and filter through a 0.45 μm syringe filter. Set aside 1 mL for a subsequent protein quantification assay and visualization by SDS-PAGE analysis. 4. Protein quantification: use bicinchoninic acid (BCA, 28) or the Bradford assay [29] for protein quantification, with bovine serum albumin (BSA) to generate a standard curve. 3.3 N-Glycoproteins Enrichment Using Lectin Cartridges
1. Equilibration step: use 10 column volumes of binding buffer (50 mL of binding buffer in the case of 5 mL chromatography cartridges) at a flow rate of 0.08 mL/min (see Note 2). 2. Loading step: samples should be loaded slowly to increase binding of the glycoproteins to the lectins (see Note 3). Collect the flow through for subsequent analysis by SDS-PAGE. 3. Washing step: wash the column with ten column volumes of binding buffer, or until the absorbance at 280 nm returns to the baseline value. 4. Elution step: elute the bound protein with five column volumes of elution buffer at a flow rate of approximately 0.75 mL/min. Collect 1 mL fractions. A typical elution chromatogram of glycoproteins is shown in Fig. 3.
3.4 N-Glycoprotein Batch Enrichment
1. Equilibration step: Mix 150 μL of Con A slurry (commercially obtained lectin resins are typically shipped as a suspension with 20 % ethanol as a preservative) with 5 mL of binding buffer in a 15 mL tubes. Briefly shake the suspension and centrifuge at 1,000 × g for 2 min. Discard the supernatant and repeat the process twice more for a total of three incubations/washes.
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2. Loading step: add the protein extract from Subheading 3.2, step 3 to the resin and shake on a rocking platform for 1 h at 4 °C. Centrifuge at 1,000 × g for 2 min and recover the supernatant for subsequent analysis by SDS PAGE. 3. Washing step: Add 15 mL of binding buffer to the Con A resin with the bound protein sample and mix thoroughly for 1 min. Centrifuge and recover supernatant/flow through. Repeat this step twice more for a total of three washes. The spectrophotometric absorbance (OD 280 nm) value can be used to approximate the protein concentration in the supernatant and used as an indication of when no more protein is being eluted from the resin. In general, three washes are sufficient to remove most of the nonspecifically bound protein, but additional washes can be used if significant amounts of proteins are still being eluted after three washes. 4. Elution step: Add 500 μL of elution buffer to the resin and mix for 1 min. Centrifuge at 1,000 × g for 2 min and recover the supernatant for analysis. Repeat this step twice more. The glycoproteins will be present in these eluted fractions. 3.5 Concentration and Dialysis
1. Pool the three fractions from the elution step and reduce the volume of the sample by 50 % in a centrifugal concentrator using a 5 kDa cutoff centrifugal concentrator (Amicon Ultra15, Millipore, Billerica MA). Next perform a solvent exchange step by first adding an equal volume of 100 mM ammonium bicarbonate (e.g., 4 mL of glycoprotein extract plus 4 mL 100 mM ammonium bicarbonate) then again reducing the volume by 50 % by centrifugation. Repeat this solvent exchange step at least three times. 2. Lyophilize the final sample containing the glycoproteins extract and resuspend the sample in 300 μL resuspension buffer, vortex for 5 min and place in a sonicating water bath for 5 min. 3. Centrifuge the suspension at 13,000 × g for 3 min. Recover the supernatant and set aside 50 μL for subsequent protein quantification and analysis by SDS-PAGE, and store the remainder at −80 °C if necessary.
3.6
Protein Digestion
1. Mix an aliquot of the sample containing 100 μg of glycoprotein with resuspension buffer to a final volume of 200 μL. 2. Add 1 μL 2 M DDT (the final concentration will be 10 mM) and incubate for 1 h at room temperature. Do not heat the samples above room temperature (see Note 4). 3. Add 20 μL 257 mM iodoacetamide (the final concentration will be 25 mM) and incubate for 30 min in the dark at room temperature.
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4. Add 1.6 mL 100 mM ammonium bicarbonate to achieve a final concentration of 0.99 M urea. 5. Add trypsin to achieve a final trypsin–glycoprotein sample ratio of 1:20 (5 μg trypsin–100 μg glycoprotein) and incubate for 16 h at 37 °C 3.7 Affinity Purification of N-Glycopeptides Using a Porous Graphitic Carbon (PGC) Column
Equilibration step: Pass the following solutions sequentially through the PGC column and discard the flow through: 1. 1 mL 1 M NaOH. 2. 2 mL water. 3. 1 mL 30 % acetic acid. 4. 2 mL water. 5. 1 mL elution solvent (50 % acetonitrile, 0.1 % formic acid [v/v] in water). 6. 1 mL wash solvent (5 % acetonitrile, 0.1 % formic acid [v/v] in water). Do not allow air to enter the column. Loading step:
1. Adjust the pH of the samples to 5.0 with 0.1 % trifluoroacetic acid (TFA). 2. Slowly load the sample (approximately 1 drop/s) onto the column followed by 1 mL water. Collect the flow through for subsequent analysis by MS if needed to evaluate the unbound peptides. Desalting step:
1. Pass 1 mL wash solvent through the column and recover the flow through for subsequent analysis by MS if needed. This sample can then be desalted using a conventional reverse phase C18 solid phase extraction prior to analysis by MS (see Note 5). Elution step:
1. Pass 1 mL elution solvent through the cartridge bed and collect the flow through. 2. Gently pass air through the column to elute all the solvent into a collection tube. 3. Dry the sample in a rotary evaporator (e.g., Savant) prior to downstream MS analysis. After the glycoprotein digestion a pool of peptides and glycopeptides will be present in the enriched samples. After the desalting and cleanup steps various downstream experiments are possible. These include protein sequence identification, characterization of glycopeptide sequences, determination of the glycosylation sites and interpretation of N-glycan structure in the N-glycopeptides.
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Notes 1. Phenylmethanesulfonylfluoride (PMSF) can be added to the protease inhibitor cocktail (1 mM final concentration) for better protease inhibition. 2. Is important to note that cartridges used for the first time can leak some of the lectin during the glycoprotein elution step. Therefore, it is recommended to wash and equilibrate the columns with more than 10 volumes of binding buffer prior to application of the sample. 3. When the volume of the sample is large and the proteins concentration is low, it may help to perform the loading step in a cold room with a peristaltic pump. It is important to load the samples at as a low a rate as possible. Alternatively, a fast protein liquid chromatography (FPLC; GE Healthcare) system with a sample loop can be used. 4. Protein samples in buffers containing urea must not be heated because this can cause carbamylation: urea in solution is in equilibrium with ammonium cyanate and the isocyanic acid reacts with protein amino groups. This results in considerable charge heterogeneity, which complicates subsequent MS analysis. 5. The majority of the glycopeptides bind to the PGC matrix; however, some peptides and glycopeptide do not bind. If an objective is to identify non-glycosylated peptides that were derived from the original glycoproteins, then analysis of the flow through may also be included at this point.
Acknowledgments Funding to JKCR for research in this area is provided by the NSF Plant Genome Research Program (DBI-0606595) and the New York State Office of Science, Technology and Academic Research (NYSTAR). References 1. Marino K, Bones J, Kattla JJ et al (2010) A systematic approach to protein glycosylation analysis: a path through the maze. Nat Chem Biol 6:713–723 2. An HJ, Froehlich JW, Lebrilla CB (2009) Determination of glycosylation sites and site-specific heterogeneity in glycoproteins. Curr Opin Chem Biol 13:421–426 3. Pless DD, Lennarz WJ (1977) Enzymatic conversion of proteins to glycoproteins. Proc Natl Acad Sci USA 74:134–138
4. Matsuoka K, Watanabe N, Nakamura K (1995) O-glycosylation of a precursor to a sweet potato vacuolar protein, sporamin, expressed in tobacco cells. Plant J 8:877–889 5. Cho YP, Chrispeels MJ (1976) SerineO-galactosyl linkages in glycopeptides from carrot cell-walls. Phytochemistry 15: 165–169 6. Showalter AM (2001) Arabinogalactan-proteins: structure, expression and function. Cell Mol Life Sci 58:1399–1417
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7. Velasquez SM, Ricardi MM, Dorosz JG et al (2011) O-glycosylated cell wall proteins are essential in root hair growth. Science 332: 1401–1403 8. Zielinska DF, Gnad F, Wisniewski JR et al (2010) Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell 141:897–907 9. Kaji H, Kamiie J, Kawakami H et al (2007) Proteomics reveals N-linked glycoprotein diversity in Caenorhabditis elegans and suggests an atypical translocation mechanism for integral membrane proteins. Mol Cell Proteomics 6:2100–2109 10. Wollscheid B, Bausch-Fluck D, Henderson C et al (2009) Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins. Nat Biotechnol 27: 378–386 11. Gundry RL, Raginski K, Tarasova Y et al (2009) The mouse C2C12 myoblast cell surface N-linked glycoproteome: identification, glycosite occupancy, and membrane orientation. Mol Cell Proteomics 8:2555–2569 12. Liu T, Qian WJ, Gritsenko MA et al (2005) Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry. J Proteome Res 4:2070–2080 13. Bunkenborg J, Pilch BJ, Podtelejnikov AV et al (2004) Screening for N-glycosylated proteins by liquid chromatography mass spectrometry. Proteomics 4:454–465 14. Lee A, Kolarich D, Haynes PA et al (2009) Rat liver membrane glycoproteome: enrichment by phase partitioning and glycoprotein capture. J Proteome Res 8:770–781 15. Minic Z, Jamet E, Negroni L et al (2007) A sub-proteome of Arabidopsis thaliana mature stems trapped on Concanavalin A is enriched in cell wall glycoside hydrolases. J Exp Bot 58:2503–2512 16. Catala C, Howe KJ, Hucko S et al (2011) Towards characterization of the glycoproteome of tomato (Solanum lycopersicum) fruit using Concanavalin A lectin affinity chromatography and LC-MALDI-MS/MS analysis. Proteomics 8:1530–1544 17. Zhang Y, Giboulot A, Zivy M et al (2010) Combining various strategies to increase the coverage of the plant cell wall glycoproteome. Phytochemistry 10:1109–1123 18. Zhang H, Li XJ, Martin DB et al (2003) Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol 21:660–666 19. Sparbier K, Koch S, Kessler I et al (2005) Selective isolation of glycoproteins and glycopeptides for MALDI-TOF MS detection
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supported by magnetic particles. J Biomol Tech 16:407–413 Nilsson CL (2011) Lectin techniques for glycoproteomics. Curr Proteomics 8:248–256 McDonald CA, Yang JY, Marathe V et al (2009) Combining results from lectin affinity chromatography and glycocapture approaches substantially improves the coverage of the glycoproteome. Mol Cell Proteomics 8: 287–301 Choi E, Loo D, Dennis JW et al (2011) High-throughput lectin magnetic bead arraycoupled tandem mass spectrometry for glycoprotein biomarker discovery. Electrophoresis 32:3564–3575 Yang G, Cui T, Chen Q et al (2012) Isolation and identification of native membrane glycoproteins from living cell by concanavalin A-magnetic particle conjugates. Anal Biochem 421:339–341 Yamamoto K, Tsuji T, Osawa T (1998) Analysis of asparagine-linked oligosaccharides by sequential lectin-affinity chromatography. Methods Mol Biol 76:35–51 Yamamoto K, Tsuji T, Osawa T (1995) Analysis of asparagine-linked oligosaccharides by sequential lectin affinity chromatography. Mol Biotechnol 3:25–36 Yang Z, Harris LE, Palmer-Toy DE et al (2006) Multilectin affinity chromatography for characterization of multiple glycoprotein biomarker candidates in serum from breast cancer patients. Clin Chem 52:1897–1905 Li L, Wang L, Zhang W et al (2004) Correlation of serum VEGF levels with clinical stage, therapy efficacy, tumor metastasis and patient survival in ovarian cancer. Anticancer Res 24:1973–1979 Smith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 Hardman KD, Ainsworth CF (1972) Myoinositol binding site of concanavalin A. Nature 237:54–55 Hardman KD, Ainsworth CF (1972) Structure of concanavalin A at 2.4-A resolution. Biochemistry 11:4910–4919 Sumner JB (1919) The globulins of the jack bean, Canavalia ensiformis. J Biol Chem 37:137–142 Sumner JB, Howell SF (1936) Identification of hemagglutinin of jack bean with concanavalin A. J Bacteriol 32:227–237 Kornfeld K, Reitman ML, Kornfeld R (1981) The carbohydrate-binding specificity of pea
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and lentil lectins. Fucose is an important determinant. J Biol Chem 256:6633–6640 Shibuya N, Goldstein IJ, Van Damme EJ et al (1988) Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb. J Biol Chem 263: 728–734 Onozaki K, Homma Y, Hashimoto T (1979) Purification of an L-fucose binding lectin from Ulex europeus by affinity column chromatography. Experientia 35:1556–1557 Kochibe N, Furukawa K (1980) Purification and properties of a novel fucose-specific hemagglutinin of Aleuria aurantia. Biochemistry 19:2841–2846 Nicolson GL, Blaustein J, Etzler ME (1974) Characterization of two plant lectins from Ricinus communis and their quantitative interaction with a murine lymphoma. Biochemistry 13:196–204 Nicolson GL, Blaustein J (1972) The interaction of Ricinus communis agglutinin with normal and tumor cell surfaces. Biochim Biophys Acta 266:543–547 Farrar GH, Uhlenbruck G, Holz G (1980) Comparison of isolated peanut agglutinin receptor glycoproteins from human, bovine and porcine erythrocyte membranes. Biochim Biophys Acta 603:185–197 Lotan R, Skutelsky E, Danon D et al (1975) The purification, composition, and specificity of the anti-T lectin from peanut (Arachis hypogaea). J Biol Chem 250:8518–8523
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42. Novogrodsky A, Lotan R, Ravid A et al (1975) Peanut agglutinin, a new mitogen that binds to galactosyl sites exposed after neuraminidase treatment. J Immunol 115:1243–1248 43. Bunn-Moreno MM, Campos-Neto A (1981) Lectin(s) extracted from seeds of Artocarpus integrifolia (jackfruit): potent and selective stimulator(s) of distinct human T and B cell functions. J Immunol 127:427–429 44. Grubhoffer L, Ticha M, Kocourek J (1981) Isolation and properties of a lectin from the seeds of hairy vetch (Vicia villosa Roth). Biochem J 195:623–626 45. Kimura A, Wigzell H, Holmquist G et al (1979) Selective affinity fractionation of murine cytotoxic T lymphocytes (CTL). Unique lectin specific binding of the CTL associated surface glycoprotein, T 145. J Exp Med 149:473–484 46. Nagata Y, Burger MM (1974) Wheat germ agglutinin. Molecular characteristics and specificity for sugar binding. J Biol Chem 249: 3116–3122 47. Nagata Y, Burger MM (1972) Wheat germ agglutinin. Isolation and crystallization. J Biol Chem 247:2248–2250 48. Shibuya N, Goldstein IJ, Broekaert WF et al (1987) The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac(alpha 2-6) Gal/GalNAc sequence. J Biol Chem 262: 1596–1601 49. Yamamoto K, Konami Y, Irimura T (1997) Sialic acid-binding motif of Maackia amurensis lectins. J Biochem 121:756–761
1
Introduction Glycosylation is a highly complex posttranslational modification associated with many eukaryotic proteins, involving the attachment of oligosaccharide moieties and their subsequent modification by a large battery of glycan modifying enzymes. This results in structurally diverse pool of glycoproteins and glycoforms [1, 2]. In plants, these glycoproteins can be classified in N-glycoproteins, where N-glycans are covalently linked to asparagine in the sequon N-X-(S/T), where X can be any amino acid except proline [3], and O-glycoproteins, where in the glycan is attached to the hydroxyl group of serine, threonine, or hydroxylated proline residues [4–7], with no specific sequon. Several analytical platforms have been developed for systematic studies of glycoproteins from bacteria, yeast and animals [8–14], but there are not yet an analogous system for plant glycoproteomes. A typical workflow might comprise front-end enrichment of glycoproteins/glycopeptides, identification of peptide sequence, determination of glycosylation sites and
Jesus V. Jorrin-Novo et al. (eds.), Plant Proteomics: Methods and Protocols, Methods in Molecular Biology, vol. 1072, DOI 10.1007/978-1-62703-631-3_43, © Springer Science+Business Media, LLC 2014
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Fig. 1 Strategy workflow for systematic study of N-glycoproteins
site occupancy, and interpretation of glycan structure and glycoforms (Fig. 1). However, several factors can complicate the glycoprotein analysis, including the complexity of biological samples and low protein abundance. Therefore, the reduction of the complexity and enrichment of glycoproteins can represent an effective first step. This can be accomplished by lectin affinity chromatography [15–17] and chemical methods, such as hydrazine chemistry [18] and boronic acid [19]. However, lectin affinity chromatography with Concanavalin A has been more commonly used for studies of plant glycoproteins [15–17] and a number of glycoproteomic analyses have been reported in the last few years employing lectin affinity as an early enrichment step [20], including those that use multiple lectins to increase the population of captured glycoproteins [17]. To this end, a range of lectins with different affinities is now commercially available (Table 1). However, the chemical methods should be considered as complementary analytical approaches for a systematic study [14, 21], although they are not further discussed here. In this chapter we present a protocol to enrich for N-glycoproteins from plant tissues using lectin affinity chromatography, and to prepare the samples for downstream experiments designed to identify the proteins using mass spectrometry (MS).
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Table 1 Commercially available glycan-binding lectins for the enrichment of glycoproteins Lectin type
Name
Source
Affinity
Mannose binding lectins
Con A (concanavalin A)
Canavalia ensiformis
High-mannose, hybrid and biantennary complex type N-glycans [30–33]
LCH (Lentil lectin)
Lens culinaris
Fucosylated core region of bi- and triantennary complex type N-glycans [34]
GNA (Snowdrop lectin)
Galanthus nivalis
α-1,3 and α-1,6 link high mannose structure [35]
UEA (Ulex europaeus agglutinin)
Ulex europaeus
Fucα1-2Gal-R [36]
AAL (Aleuria aurantia)
Aleuria aurantia
Fucα1-2Galβ1-4(Fucα13/4) Galβ1-4GlcNAc; R2GlcNAcβ1-4(Fucα1 6) GlcNAc-R1 [37]
RCA (Ricinus communis Agglutinin)
Ricinus communis
Galβ1-4GlcNAcβ1-R [38, 39]
PNA (Peanut Agglutinin)
Arachis hypogaea
Galβ1-3GalNAcα1-Ser/Thr (T-Antigen) [40–42]
AIL (Jacalin)
Artocarpus integrifolia
(Sia)Galβ1-3GalNAcα1-Ser/ Thr (T-Antigen) [43]
VVL (Hairy vetch lectin)
Vicia villosa
GalNAcα-Ser/Thr (Tn-Antigen) [44, 45]
WGA (Wheat Germ agglutinin)
Triticum vulgaris
GlcNAcβ1-4GlcNAcβ14GlcNAc, Neu5Ac (sialic acid) [46, 47]
SNA (Elderberry lectin)
Sambucus nigra
Neu5Acα2-6Gal(NAc)-R [48]
MAL (Maackia amurensis lectin)
Maackia amurensis
Neu5Ac/Gcα2-3Galβ14GlcNAcβ1-R [49]
Fucose binding lectins
Galactose/Nacetylgalactosamine binding lectins
Sialic acid/Nacetylglucosamine binding lectins
2
Materials Note: mention of specific companies or pieces of equipment does not represent an endorsement by the authors.
2.1
Lectin Resins
Several lectins coupled to Sepharose beads, magnetic beads [22, 23] or to beads packed into chromatography columns/cartridges are commercially available (e.g., Qiagen) to facilitate the enrichment of a diverse population of glycoproteins (Table 1). Of these, Concanavalin A (Con A) is the most frequently used. It is possible to work with several lectins sequentially [24, 25], in parallel [8, 14, 26]
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Fig. 2 Different modalities of lectin affinity chromatography. Lectin enrichment can be done in parallel (a), sequentially (b), and using a mix of several lectins (c). The type of lectins used can be tailored to the kind of glycoprotein that is being targeted
and as mixtures [27], as illustrated in Fig. 2. If the main goal is to enrich for N-glycoproteins an effective option is the use of mannose binding lectins (Table 1). 2.2 Porous Graphitic Carbon (PGC) Columns
Prepacked PGC columns (1 mL, brand name Hypersep Hypercarb) can be purchased from Thermo Scientific.
2.3
Buffer solutions must be prepared with bi-distilled water, filtered with 0.45 μm filters, degassed, and precooled to 4 °C before starting the main protocol.
Buffer Solutions
1. Stock buffers: prepare 1 M Tris, pH 7.0 (Solution A) and 5 M NaCl (Solution B) solutions. Dissolve 121.4 g Tris in 900 mL water, and adjust the pH with HCl, and the final volume to 1 L with water. Dissolve 292 g NaCl in 500 mL water and adjust the volume to 1 L. 2. Protein Extraction buffer: 25 mM Tris, pH 7.0, 0.5 M NaCl, 0.2 M CaCl2, and 20 μL/g fresh weight protease inhibitor cocktails. For 100 mL of protein extraction buffer mix: 2.5 mL A solution, 10 mL B solution, 2.94 g CaCl2, and 20 μL/g fresh weight protease inhibitor cocktails and adjust the volume to 100 mL (see Note 1). 3. Binding buffer (Lectin): 20 mM Tris–HCl, 0.5 M NaCl, 1 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2. Mix 2 mL Solution
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A, 10 mL Solution B, 0.0147 g CaCl2, 0.0197 g MnCl2, and 0.0203 g MgCl2 and adjust the volume to 100 mL. 4. Elution buffer (Lectin): binding buffer plus 0.5 M α-methyl-Dmannopyranoside. Dissolve 9.71 g α-methyl-D-mannopyranoside in 100 mL binding buffer. 5. 100 mM Ammonium bicarbonate (Na2CO3): dissolve 1.05 g Na2CO3 in water and adjust the final volume to 100 mL. 6. Resuspension buffer: 8 M urea, 100 mM Na2CO3. Dissolve 0.961 g urea in 2 mL 100 mM Na2CO3. Prepare this solution fresh immediately prior to use. 7. 2 M Dithiothreitol (DTT): dissolve 0.154 g DTT in 100 mM Na2CO3, adjust the final volume to 500 μL, divide into small aliquots (e.g., 5 μL) and store at −80 °C until use. 8. 257 mM Iodoacetamide: dissolve 0.023 g iodoacetamide in 100 mM Na2CO3 and adjust the final volume 500 μL. Prepare this solution fresh immediately prior to use. 9. 1 M NaOH: dissolve 4 g NaOH in water and adjust the volume to 100 mL. 10. 30 % Acetic acid: mix 30 mL glacial acetic acid with 70 mL water. This solution should be prepared in a fume hood to avoid potentially toxic fumes. 11. Porous graphitic Carbon (PGC) wash solvent: 5 % acetonitrile, 0.1 % formic acid (v/v) in water. Mix 5 mL acetonitrile and 100 μL formic acid and adjust the volume to 100 mL with water. This solution should be prepared in the fume hood. 12. PGC Elution solvent: 50 % acetonitrile, 0.1 % formic acid (v/v) in water. Mix 50 mL and 100 μL formic acid and adjust the volume to 100 mL with water. This solution should be prepared in the fume hood.
3 3.1
Methods Tissue Collection
3.2 Protein Extraction
1. Weigh out 3 g of plant material (fresh or flash frozen in liquid nitrogen and stored at −80 °C). Replicated biological samples should also be considered, depending on the experimental goals. 1. Powder the samples in liquid nitrogen using a pestle and mortar and add a tenth mass of polyvinylpolypyrrolidone (PVPP; 1 g/10 g fresh weight) to help remove phenolic compounds. 2. Homogenize the material in three volumes of extraction buffer (45 mL) with a tissue homogenizer (e.g., Polytron, Kinematica) for 15 s, and then filter with Miracloth (Calbiochem). Shake the crude extract at 5 rpm on a rocking platform for 2 h at 4 °C.
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Fig. 3 A typical UV absorbance chromatogram of the N-glycoprotein enriched fraction from a crude protein extract
3. Centrifuge the crude extract at 15,000 × g for 30 min. Recover the supernatant and filter through a 0.45 μm syringe filter. Set aside 1 mL for a subsequent protein quantification assay and visualization by SDS-PAGE analysis. 4. Protein quantification: use bicinchoninic acid (BCA, 28) or the Bradford assay [29] for protein quantification, with bovine serum albumin (BSA) to generate a standard curve. 3.3 N-Glycoproteins Enrichment Using Lectin Cartridges
1. Equilibration step: use 10 column volumes of binding buffer (50 mL of binding buffer in the case of 5 mL chromatography cartridges) at a flow rate of 0.08 mL/min (see Note 2). 2. Loading step: samples should be loaded slowly to increase binding of the glycoproteins to the lectins (see Note 3). Collect the flow through for subsequent analysis by SDS-PAGE. 3. Washing step: wash the column with ten column volumes of binding buffer, or until the absorbance at 280 nm returns to the baseline value. 4. Elution step: elute the bound protein with five column volumes of elution buffer at a flow rate of approximately 0.75 mL/min. Collect 1 mL fractions. A typical elution chromatogram of glycoproteins is shown in Fig. 3.
3.4 N-Glycoprotein Batch Enrichment
1. Equilibration step: Mix 150 μL of Con A slurry (commercially obtained lectin resins are typically shipped as a suspension with 20 % ethanol as a preservative) with 5 mL of binding buffer in a 15 mL tubes. Briefly shake the suspension and centrifuge at 1,000 × g for 2 min. Discard the supernatant and repeat the process twice more for a total of three incubations/washes.
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2. Loading step: add the protein extract from Subheading 3.2, step 3 to the resin and shake on a rocking platform for 1 h at 4 °C. Centrifuge at 1,000 × g for 2 min and recover the supernatant for subsequent analysis by SDS PAGE. 3. Washing step: Add 15 mL of binding buffer to the Con A resin with the bound protein sample and mix thoroughly for 1 min. Centrifuge and recover supernatant/flow through. Repeat this step twice more for a total of three washes. The spectrophotometric absorbance (OD 280 nm) value can be used to approximate the protein concentration in the supernatant and used as an indication of when no more protein is being eluted from the resin. In general, three washes are sufficient to remove most of the nonspecifically bound protein, but additional washes can be used if significant amounts of proteins are still being eluted after three washes. 4. Elution step: Add 500 μL of elution buffer to the resin and mix for 1 min. Centrifuge at 1,000 × g for 2 min and recover the supernatant for analysis. Repeat this step twice more. The glycoproteins will be present in these eluted fractions. 3.5 Concentration and Dialysis
1. Pool the three fractions from the elution step and reduce the volume of the sample by 50 % in a centrifugal concentrator using a 5 kDa cutoff centrifugal concentrator (Amicon Ultra15, Millipore, Billerica MA). Next perform a solvent exchange step by first adding an equal volume of 100 mM ammonium bicarbonate (e.g., 4 mL of glycoprotein extract plus 4 mL 100 mM ammonium bicarbonate) then again reducing the volume by 50 % by centrifugation. Repeat this solvent exchange step at least three times. 2. Lyophilize the final sample containing the glycoproteins extract and resuspend the sample in 300 μL resuspension buffer, vortex for 5 min and place in a sonicating water bath for 5 min. 3. Centrifuge the suspension at 13,000 × g for 3 min. Recover the supernatant and set aside 50 μL for subsequent protein quantification and analysis by SDS-PAGE, and store the remainder at −80 °C if necessary.
3.6
Protein Digestion
1. Mix an aliquot of the sample containing 100 μg of glycoprotein with resuspension buffer to a final volume of 200 μL. 2. Add 1 μL 2 M DDT (the final concentration will be 10 mM) and incubate for 1 h at room temperature. Do not heat the samples above room temperature (see Note 4). 3. Add 20 μL 257 mM iodoacetamide (the final concentration will be 25 mM) and incubate for 30 min in the dark at room temperature.
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4. Add 1.6 mL 100 mM ammonium bicarbonate to achieve a final concentration of 0.99 M urea. 5. Add trypsin to achieve a final trypsin–glycoprotein sample ratio of 1:20 (5 μg trypsin–100 μg glycoprotein) and incubate for 16 h at 37 °C 3.7 Affinity Purification of N-Glycopeptides Using a Porous Graphitic Carbon (PGC) Column
Equilibration step: Pass the following solutions sequentially through the PGC column and discard the flow through: 1. 1 mL 1 M NaOH. 2. 2 mL water. 3. 1 mL 30 % acetic acid. 4. 2 mL water. 5. 1 mL elution solvent (50 % acetonitrile, 0.1 % formic acid [v/v] in water). 6. 1 mL wash solvent (5 % acetonitrile, 0.1 % formic acid [v/v] in water). Do not allow air to enter the column. Loading step:
1. Adjust the pH of the samples to 5.0 with 0.1 % trifluoroacetic acid (TFA). 2. Slowly load the sample (approximately 1 drop/s) onto the column followed by 1 mL water. Collect the flow through for subsequent analysis by MS if needed to evaluate the unbound peptides. Desalting step:
1. Pass 1 mL wash solvent through the column and recover the flow through for subsequent analysis by MS if needed. This sample can then be desalted using a conventional reverse phase C18 solid phase extraction prior to analysis by MS (see Note 5). Elution step:
1. Pass 1 mL elution solvent through the cartridge bed and collect the flow through. 2. Gently pass air through the column to elute all the solvent into a collection tube. 3. Dry the sample in a rotary evaporator (e.g., Savant) prior to downstream MS analysis. After the glycoprotein digestion a pool of peptides and glycopeptides will be present in the enriched samples. After the desalting and cleanup steps various downstream experiments are possible. These include protein sequence identification, characterization of glycopeptide sequences, determination of the glycosylation sites and interpretation of N-glycan structure in the N-glycopeptides.
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Notes 1. Phenylmethanesulfonylfluoride (PMSF) can be added to the protease inhibitor cocktail (1 mM final concentration) for better protease inhibition. 2. Is important to note that cartridges used for the first time can leak some of the lectin during the glycoprotein elution step. Therefore, it is recommended to wash and equilibrate the columns with more than 10 volumes of binding buffer prior to application of the sample. 3. When the volume of the sample is large and the proteins concentration is low, it may help to perform the loading step in a cold room with a peristaltic pump. It is important to load the samples at as a low a rate as possible. Alternatively, a fast protein liquid chromatography (FPLC; GE Healthcare) system with a sample loop can be used. 4. Protein samples in buffers containing urea must not be heated because this can cause carbamylation: urea in solution is in equilibrium with ammonium cyanate and the isocyanic acid reacts with protein amino groups. This results in considerable charge heterogeneity, which complicates subsequent MS analysis. 5. The majority of the glycopeptides bind to the PGC matrix; however, some peptides and glycopeptide do not bind. If an objective is to identify non-glycosylated peptides that were derived from the original glycoproteins, then analysis of the flow through may also be included at this point.
Acknowledgments Funding to JKCR for research in this area is provided by the NSF Plant Genome Research Program (DBI-0606595) and the New York State Office of Science, Technology and Academic Research (NYSTAR). References 1. Marino K, Bones J, Kattla JJ et al (2010) A systematic approach to protein glycosylation analysis: a path through the maze. Nat Chem Biol 6:713–723 2. An HJ, Froehlich JW, Lebrilla CB (2009) Determination of glycosylation sites and site-specific heterogeneity in glycoproteins. Curr Opin Chem Biol 13:421–426 3. Pless DD, Lennarz WJ (1977) Enzymatic conversion of proteins to glycoproteins. Proc Natl Acad Sci USA 74:134–138
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