Plant proteomics and glycosylation experiments
Like other eukaryotic cells, glycosylation is one of the most studied post-translational events in plant cells. Depending on the type of connection between the oligosaccharide fraction and the protein backbone, glycosylation can be divided into two types: N-glycosylation and O-glycosylation. The source of this experiment is the "Guide to Plant Proteomics Experiments" [French] H. Tillemment, M. Zivi, C. Damerweil, V. Mitchen, eds.
Operation method
Plant proteomics and glycosylation
Materials and Instruments
Streptavidin-Peroxidase Bovine Pancreatic Ribonuclease B Methyl Bimanual Glycoside Ovalbumin Move 3.1 Is this protein a glycoprotein? For more product details, please visit Aladdin Scientific website.
DIG Sugar Chain Reagent TTBS buffer Lectin- biotin TBS buffer
There is only one way to fully answer the question, "Is this protein a glycoprotein?" There is only one way to fully answer the question "Is this protein a glycoprotein? This requires the use of a total glycan assay kit that can detect and quantify glycoproteins on the blot. The Roche Applied Science commercial ----DIG Glycan Assay Kit (instructions for use included with the product) provides the reagents needed for these assays. It should be noted that when using this kit, proteins are bound to at least one glycosidic portion in order to obtain experimental results, and these assays do not provide information about the type of oligosaccharide molecules involved in glycosylation and their attachment to the protein backbone.
3.2 How proteins are glycosylated
There are several methods that can be used for the characterization of glycosides on glycoproteins. One method consists of Western blotting experiments applying probes specific for the oligosaccharide portion of glycoproteins, which are available in both lectin-type and antibody-type probes. This method requires neither purification of the glycoprotein sample nor dissociation or isolation of the glycosides (see 25. 3. 2 Section 1.1). Another method involves isolating the glycoprotein and directly analyzing the monosaccharide composition of the glycosides attached to the glycoprotein (see 25. 3. 2 Section 2.1). A further method is the selective dissociation of glycosides from purified glycoproteins by chemical or enzymatic treatment. The glycoproteins are then analyzed by electrophoresis or mass spectrometry before or after deglycosylation (see 25. 3. 2, section 2.1). Selective cleavage brings us to the characterization of the glycosides attached to the glycoprotein. At the same time, differences in the molecular mass of the proteins identified by mass spectrometry will provide us with structural information about the glycosides attached to the glycoproteins.
1. Western blotting for detection of glycosides
Western blotting of one- or two-dimensional electrophoresis gels can be used to detect glycosides on glycoproteins. Most of the probes used for this assay are N-conjugated glycoside specific.
1 ) Lectin affinity assay
Lectins, a class of plant proteins that bind specifically to oligosaccharide fractions, have been identified with affinity for mammalian glycoproteins and are listed in Table 25-1. Only a small number of these lectins can be used for the detection of plant glycoproteins (see Note 2).
These lectin commodities are lectin-biotinylated forms. Lectins and oligosaccharides are recognized by the streptavidin-horseradish peroxidase marker. However, the only exception is cutinoglobulin A, which binds directly to peroxidase. Below are the specifics of these two programs:
Lectin- Biotin/Streptavidin-Peroxidase method:
( 1 ) Separated by 1D or 2D electrophoresis and transferred to a nitrocellulose membrane.
( 2 ) Soak the blotting membrane with TTBS buffer for 1 h (see Note 3).
( 3 ) Soak the blotting membrane in TTBS buffer containing lectin-biotin complex (0.1 mg/20 ml) for 2 h at room temperature.
( 4 ) Rinse the membrane in TTBS buffer 4 times for 15 min each time.
( 5 ) Place the blotting membrane in streptavidin-horseradish peroxidase standard diluted 1:1000 in TTBS buffer and incubate for 1 h at room temperature.
( 6 ) Rinse the blotting membrane in TTBS buffer 4 times (15 min each time), and rinse it once in TBS solution before developing.
( 7 ) Dissolve 30 mg of 4-chloro-1-naphthol in 10 ml of cold methanol (1-20°C) in one beaker, and add 30 μl of H2O2 to 50 ml of TBS buffer in the other beaker, and when it is time to develop the blotting membrane, combine the solutions from the above two beakers to make a peroxidase development mix (to be prepared as it is used), and shake gently to optimize the development reaction.
( 8 ) Pour off the development mixture and rinse the blotting film several times with distilled water to terminate the development reaction. Dry the blotting film and store it between two layers of filter paper.
( 9 ) For experimental control, see Note 4.
Knife Bean Globulin A (ConA) - Peroxidase Method (method modified from Ref. 20).
( 1 ) Separated by 1D or 2D electrophoresis and transferred to a nitrocellulose membrane.
( 2 ) Soak the blotting membrane with TTBS buffer for 1 h. The blotting membrane was then blotted with TTBS buffer.
( 3 ) The blotting membrane was incubated in TTBS buffer containing ConA ( 25 μg/ml) for 2 h at room temperature.
( 4 ) Rinse the blotting membrane in TTBS buffer four times, each time for 15 min.
( 5 ) Place the membrane in TTBS buffer containing horseradish peroxidase (HRP, 50 μg/mL) and incubate for 1 h at room temperature.
( 6 ) Rinse the blotting membrane in TTBS buffer 4 times (15 min each time), and rinse it in TBS solution once before developing.
( 7 ) Peroxidase development was performed as in the lectin-biotin/streptavidin-horseradish peroxidase method.
( 8 ) The experiment must be controlled, see Note 4.
2 ) Immunoassay for specific antibodies
We have reported that the β-1-2 xylose and α-1-3-fucose antigenic epitopes of plant N-complex glycosides are highly immunogenic in rabbit serum [21] . Thus, glycoprotein antisera containing complex glycosides often contain antibodies against β-1-2 pentose and α-1-3 fucose. Some commercial immune sera have the same properties as our homemade probes. For example, an anti-HRP immune serum can be used as a specific probe for the complex N-glycosides of plants containing α-1-3 fucose and β-1-2 xylose residues. Immune sera obtained from bee venom proteins are more specific and can be used as specific probes for plant complex glycosides containing α-1-3 fucose [ 21] . In our laboratory, rabbit antibodies specific for the complex glycoside-terminated antenna (trisaccharide Lewisa ) were obtained from rabbit serum [ 22] . Monoclonal antibodies against Lewis a are commercially available, but they are very expensive. 
Arabinogalactan protein (AGP ) and stretch proteins can be detected with commercial monoclonal antibodies (http://W W W . plantprobes.co.uk/) . These antibodies have not been used in our laboratory; please refer to the original paper describing the specificity of these antibodies (see Note 5).
( 1 ) Separated by 1D or 2D electrophoresis and transferred to a nitrocellulose membrane.
( 2 ) Soak the blotting membrane with TBS buffer containing 3% gelatin for 1 h at room temperature.
( 3 ) Soak the blotting membrane for 2 h at room temperature in TBS buffer containing 1% gelatin and the appropriate dilution of immune serum. 25. (Section 2.2 1.2) We use the commercial immune serum referred to in Section 1.2), diluted as follows:
a. Rabbit anti-HRP immune serum, 1 : 300.
b. Rabbit anti-bee venom protein immune serum, 1 : 200.
c. Mouse anti-Lewis monoclonal antibody, 1 : 100.
( 4 ) Rinse 4 times in TTBS buffer for 15 min each time.
( 5 ) Soak the blotting membrane in TBS buffer containing 1% gelatin and appropriate labeled antibody at room temperature for 1 h. The labeled antibody can be HRP-labeled goat anti-rabbit IgG (G-type immunoglobulin) antibody at a dilution of 1 : 2000 or HRP-labeled goat anti-mouse polyvalent immunoglobulin antibody at a dilution of 1 : 500.
( 6 ) Rinse the blotting membrane 4 times in TTBS buffer (15 min each time), and rinse it in TBS solution before developing.
( 7 ) Peroxidase development is the same as in the lectin-biotin/horseradish peroxidase method (see Section 25.3.2, 1.1).
( 8 ) See Notes 6 and 7 for controls.
2. Analysis of glycosidic monosaccharide fractions
Glycoside monosaccharide fractions are analyzed by hydrolysis of glycoproteins with methanol-HCl solution followed by gas chromatography to identify and analyze the monosaccharide products and their derivatives. The results of the monosaccharide fraction analysis provide preliminary data on the type of glycoside attached to the glycoprotein and/or N-linked oligosaccharides).
( 1 ) A 1 mg sample of purified protein is lyophilized in a glass tube with a PTFE-coated screw cap (see Note 8).
( 2 ) Add 5 to 10 μl of 2 mmol/L inositol reservoir solution to the protein sample to be tested (see Note 9). Lyophilize the sample again before the methanolysis reaction.
( 3 ) Add 500 μl of a 1 mol/L methanol-HCl solution to the protein sample, tighten the lid, and heat at 80°C overnight.
( 4 ) After the methanol decomposition reaction, heat dry the sample at 40°C under nitrogen protection. Note: Because methanol is toxic, it should be operated in a fume hood.
( 5 ) Rinse with 250 μl methanol and dry the treated sample under nitrogen as in step (4 ), repeat the rinsing step twice.
( 6 ) Resuspend the sample with 250 μl of methanol, followed by 25 μl of acetic acid, 25 μl of pyridine, homogenize, and leave at room temperature for 6 h. Dry the sample under nitrogen as in step (4 ).
( 7 ) Add 250 μl of silylation reagent, incubate at 80°C for 20 min ( see Note 1 ), air blow dry the sample.
( 8 ) Rinse with 1 ml of cyclohexane, air-dry and resuspend in 200 μl of cyclohexane, homogenize, centrifuge, and transfer 100 μl of the derivatized sample to a reaction vial with a cap.
( 9 ) Set the helium pressure of the gas chromatograph to 1.4 bar, and heat the syringe and FID detector to 250°C and 280°C, respectively, before injecting the sample. Set the helium pressure to 20 psi and the flow rate to 3 ml/min. Equilibrate the column temperature to 120°C before injecting 5 μl of the derivative sample (equivalent to 25 μg of protein). 280°C. ⑤ 280°C for 3 min.
3. Protein analysis after release of sugar groups
1 ) Chemical treatment of glycosyl release
Reductive amination countercuts should selectively dissociate O-glycosides from glycoproteins (see Note 10). Deglycosylated proteins are analyzed by 1D SDS-PAGE and their migration is compared with that of their glycosylated forms; an increase in electrophoretic mobility is evidence that O-linked glycosides are present on the protein (see Note 11).
( 1 ) A 1 mg sample of purified protein was lyophilized in a glass tube with a PTFE-coated screw cap.
( 2 ) Dissolve the dried sample in 500 μl of sodium borohydride solution, cover the glass tube tightly, and leave overnight at 37°C.
( 3 ) Terminate the reductive amination reaction by adding acetic acid dropwise until no gas is produced. Add 500 μl of 10% methanol solution of acetic acid. Air dry in a fume hood. Repeat the washing process 3 times to remove the remaining borate.
( 4 ) After deglycosylation, the deglycosylated proteins are precipitated with 4 times the volume of ethanol at -20°C overnight, and the precipitate is solubilized in a suitable buffer for 1D SDS-PAGE. glycosides in the ethanol phase can be recovered for analysis of the monosaccharide fractions (see Section 25.3.2 2).
2) Enzymatic treatment with N- or O-glycosidase
Treatment of purified glycoproteins with glycosidases provides further information. Endoglycosidase H ( Endo H ) can only dissociate high-mannose-type N-glycosides from plant glycoproteins by hydrolyzing the glycosidic bond between the two GlcNAc residues in the N-glycoside center (Fig. 25-3). Peptide N-glycosidase (PNGase) dissociates high-mannose N-glycosides and complex N-glycosides by hydrolyzing the bond located between the peptide backbone asparagine and the oligosaccharides proximal to the GlcNAc.PNGaseF, a N-glycosidase widely used in mammalian glycoprotein analysis, dissociates high-mannose N-glycosides and complex N-glycosides, but not the α-1-3-Glycosides attached to the neighboring GlcNAc. PNGaseA dissociates all types of plant glycosides (Fig. 25-3), but it acts almost exclusively on glycopeptides, so enzymatic hydrolysis of glycoproteins prior to deglycosylation is required [23, 24]. Glycoproteins or peptides are analyzed for deglycosylation by (i) increased electrophoretic migration, (ii) loss of immunoreactivity of glycoside-specific probes for glycoproteins after Endo H or PNGaseF treatment, and (iii) mass spectrometry after EndoH, PNGase F, or PNGase A treatment. The same means and methods can be used for O-glycosidases, but they are rarely used in our laboratory and are therefore not described in detail here (see Refs. 25 and 26 for details).
Endoglycosidase H deglycosylation.
( 1 ) Prior to enzymatic treatment with EndoH deglycosylation, the protein obtained by purification was heat denatured at 100°C for 5 min in the presence of 1% SDS ( m/V). 
( 2 ) Dilute the sample 5-fold with 150 mmol/L sodium acetate solution (pH 5.7), add 10 mU of Endo H, and incubate the mixture at 37°C for 6 h. The sample was then incubated for 5 h at 37°C.
( 3 ) If the digest is to be used for electrophoresis, affinity reaction or immunoassay, add an equal volume of twice the concentration of electrophoresis sample buffer after the deglycosylation reaction (see Section 25.3.2.1), and desalinate the digested samples for mass spectrometry.
Peptide N-glycosidase F deglycosylation:
( 1 ) Digest the protein samples to be digested with 0.1 mmol/L Tris-HCl (pH 7.5) containing 0.1% SDS.
( 2 ) Heat the sample at 100℃ for 5 min to denature the protein. Cool down at room temperature and add an equal volume of 0.1 mmol/L Tris-HCl containing 0.5% Nonidet P-40, pH 7.5 (see Note 12).
( 3 ) The samples were incubated with peptide N-glycosidase F at 37°C for 24 h ( 1 U enzyme/100 μg protein).
( 4 ) After enzymatic digestion, precipitate the deglycosylated proteins with 4-fold volume of ethanol at -20°C, overnight.
( 5 ) Centrifuge the sample. Recover the deglycosylated proteins from the precipitation and dissolve them in appropriate buffer for gel electrophoresis and mass spectrometry.
Peptide N-glycosidase A deglycosylation:
( 1 ) Dissolve 100 μg of protein sample in 500 μl of 10 mmol/L HCl ( pH 2.2 ), add 10 μg of pepsin, digest for 24 h at 37°C, add the same amount of pepsin again, and continue to digest for another 24 h. Terminate the reaction by heating at 100°C for 5 min.
( 2 ) Cool the sample, and then take 10% of the solution to purify the peptide and glycopeptide mixture by C18 chromatography column.
( 3 ) Rinse the C18 column with 5 mL of acetonitrile, and then rinse the C18 column with 5 mL of water. Dilute the sample with distilled water to a final volume of 1 ml and add the sample to the C18 column. The column was rinsed with 5 mL of water to remove salts from the sample and 5 ml of acetonitrile was used to elute the peptides bound to the column. The peptide sample was concentrated by blow-drying the acetonitrile. The molecular masses of the peptides and glycopeptides were determined by MALDI-TOF mass spectrometry, which allowed for the acquisition of glycopeptide mass data prior to endoglycosidase A treatment.
( 4 ) The remaining sample was freeze-dried and dissolved in 500 μl of 100 mmol/L sodium acetate solution (pH 5.5).
( 5 ) The sample was digested with 0.1 mU of peptide N-glycosidase A at 37°C for 18 h. The sample was then freeze-dried and the peptides were separated from the oligosaccharides using a C18 column as in steps (2) and (3). The deglycosylated peptides were analyzed by MALDI-TOF mass spectrometry, comparing the two spectral patterns obtained before and after deglycosylation, to obtain some ionic mass differences due to the removal of N-glycosides, and to determine the types and structural forms of the N-glycosides on the proteins by combining with the information of the enzymes used in the process of deglycosylation and the plant glycoproteins.
The reagents required for this experiment are included in the assay kit, Commercial Enzymatic Deglycosylation Kit manufactured by Prozyme, Inc. It should be noted that the kit is used according to the manufacturer's instructions. The experimenter can obtain information about the type of glycosides (N- and O- linked) on the tested glycoprotein.
3.3 Where are my glycosylated proteins?
This method allows the experimenter to obtain information about the distribution of glycosides on the protein backbone and the structure of the glycosides themselves. Such experiments can be performed on purified glycoproteins or proteins isolated from 1D or 2D electrophoresis gels. First, the proteins are digested with protein endonucleases, and the digested peptides and glycopeptide mixtures are separated by high-performance liquid chromatography (HPLC). Glycoside-containing collection fractions were analyzed for sugar fractions (see Section 25.3.2 2), followed by MALDI-TOF mass spectrometry of the glycopeptide fractions before and after peptide N-glycosidase A treatment, respectively [see Section 25.3.2 3)].
( 1 ) Dissolve 1 mg of the purified protein sample in 500 μl of 50 mmol/L ammonium bicarbonate solution (pH 8.0) and heat at 100°C for 3 min.
( 2 ) Add 50 μg of TPCK-treated trypsin and treat at 37°C for 2 h. The sample was then heated for 3 min at 100°C.
( 3 ) Add 50 μg TPCK-treated trypsin again and treat at 37°C for 2 h.
( 4 ) If double digestion is performed, dissolve 50 μg of TPCK-treated chymotrypsin in 50 mmol/L ammonium bicarbonate solution (pH 8.0) and add it to the trypsin digest. process at 37°C for 2h.
( 5 ) Heat at 100°C for 5 min to terminate the protease digestion reaction.
( 6 ) Peptides and glycopeptides from trypsin/chymotrypsin digestion were separated by reverse HPLC chromatography (C18 column) using a 60 min linear gradient elution of 0-60% B solution at a flow rate of 1 ml/min, with the peaks detected at 214 nm UV. A solution: water/acetonitrile (90:10) solution containing 0.1% TFA (trifluoroacetic acid). Solution A: Water/acetonitrile (90:10) solution containing 0.1% TFA (trifluoroacetic acid). Collect the components in a volume of 2 ml each and freeze-dry the collected components.
(7) A 10% sample of each collected fraction is analyzed for sugar fractions (see Section 25.3.2.2), and fractions containing oligosaccharides are selected.
( 8 ) Analyze the HPLC fractions containing glycopeptides by MALDI-TOF mass spectrometry to determine the mass of glycopeptides.
( 9) Digestion of the glycopeptides with peptide N-glycosidase A [see Section 25.3.2, 3.2)]. Peptides and oligosaccharides are separated on a C18 column as described previously [see Section 25.3.2 3.2)].
( 10 ) Analyze the deglycosylated peptides by MALDI-TOF mass spectrometry to determine their mass. These mass data help the experimenter to identify the glycosylation sites of the protein under test. The difference in the masses of the detected glycosylated and deglycosylated peptides will provide us with structural information about the glycosides on the glycoproteins (Table 25-2).
This experimental method can also be applied to proteins of unknown sequence. In this case, the amino acid sequence of the glycosylated peptide is analyzed and determined by LC-MS/MS ( liquid-phase tandem mass spectrometry). The dissociated glycosides can also be purified and identified by MALDI-TOF mass spectrometry using the specific process described previously [18, 19].
3.4 How to analyze the whole glycoproteome
The identification of glycoproteins in plant extracts can determine the following: (1) Which genes encode glycoproteins. (i) Which genes encode glycoproteins. (ii) Which sites on potential glycosylation sites are actually glycosylated. (iii) What are the properties and structures of the glycosides on the glycoproteins. These research processes are now known as glycoproteomics. These identification experiments have been realized in animal cells, but have not been reported for plant glycoproteome identification.
Strategies recently developed in our laboratory to study the plant glycoproteome rely on purification of immobilized lectins, separation by 1D or 2D electrophoresis, and identification by mass spectrometry. The glycoproteome has been isolated by two methods: purification of secreted glycoproteins containing high-mannose-type N-glycosides and purification of O-GlcNAc glycosylated modified glycoproteins. Only the methods of glycoprotein selection are emphasized here, not specifically electrophoretic and mass spectrometric identification. Detailed information on these techniques can be found in other chapters of this book.
1. Identification of high-mannose-type N-glycoside-containing glycoproteins
This method was established in our laboratory using rapeseed as experimental material, and this method is also applicable to other plant materials. 
( 1 ) 6 g of plant material was placed in a 4°C pre-cooled mortar and pestle, 50 ml of pre-cooled TBS buffer was added, and the proteins were extracted by grinding (see Note 13), followed by centrifugation of the extract at 10,000 g for 30 min to remove insoluble matter, and then centrifugation at 150,000 g for 1 h to precipitate the membrane fragments. The concentration of soluble protein in the supernatant is determined by the Bradford protein assay. The samples were partitioned into 20 mg of protein per portion and frozen for storage.
( 2 ) Thaw a 20 mg sample at 4°C, centrifuge at 10,000 g for 30 min, and filter through a 0.20 μm membrane to remove all insoluble material. Add cold TBS buffer to 35 ml, and add CaCl2 and MgCl2 to a final concentration of 1 mmol/L. The sample is then diluted to a final concentration of 1 ml.
( 3 ) Resin equivalent to 1 ml of sabin A-agarose resin was resuspended in 50 ml of TBS buffer, and the resin was rinsed twice with 50 ml of TBS buffer, and the resin was recovered by sedimentation or centrifugation (3500 g, 10 min) after each rinse.
( 4 ) The washed resin was shaken with 35 ml of sample on a rotary shaker at 4°C for 2 h. The resin was poured into a PolyPrep column and rinsed to remove unbound proteins.
( 5 ) Rinse the resin 5 times with 50 ml TBS buffer, then rinse the resin 5 times with 50 ml TBS buffer, and then resuspend the resin with 50 ml TBS buffer as described in step (3 ).
( 6 ) Pour the resin into the Poly-Prep column, remove the residual TBS buffer, and resuspend the resin in the column with 10 ml of TBS buffer containing 0.3 mol/L α-methylmannose. Place the column at 4°C with rotary shaking for 1 h (see Note 14).
( 7 ) Elute the glycoprotein with 15 times the volume of TBS buffer containing 0.3 mol/L α-methylmannose, rinse the resin with the same solution, and mix the two portions of the sample; the final mixture is the purified glycoprotein attached to the high mannose-type N-glycoside.
( 8 ) The protein concentration was determined using the Bradford protein assay, and the glycoprotein sample was precipitated overnight at 4°C in 12.5% trichloroacetic acid (final concentration). 10,000 g was centrifuged for 15 min to precipitate the proteins, and the precipitates were rinsed with an acetone/water [ 9 : 1 ( V/V ) ] solution and centrifuged again. The washing process is repeated twice and the proteins are dissolved in appropriate sample buffer for 2D electrophoresis analysis.
( 9 ) Glycoproteins were separated by 2D gels and protein spots were stained by gelatin blue. Collect each visualized spot separately (see Note 15). Trypsinize the collected protein spots. Analyze the enzyme digestion products by LC-MS/MS. Submit the obtained peptide sequence information to the NCBI nonredundant database for "short, almost exact match" blast searches within selectable species limits (http://www. ncbi. nlm. nih. gov/BLAST/).
Once identified, glycoproteins should be identified in two ways: ① Are they secreted, or do they have a potential signal peptide? (ii) Do they possess at least one potential N-glycosylation site? Glycosylation sites in a protein can be identified by comparison with the Prosite database (http : //npsa- pbil. ibcp . fr /cgibin / npsa _ autom at, p i ? page = npsa _ prosite , html or http : // www . expasy . org /tools /scanprosite / ) Compare to determine.
2. Identification of O-site N-acetylglucosamine glycosylated proteins
Identification of the O-GlcNAc glycoproteome requires glycoprotein purification by wheat germ agglutinin (WGA) affinity chromatography. Previous experiments have shown that WGA binds both O-GlcNAc glycoproteins and proteins with N-glycosides [ 28]. Therefore, the N-glycosides must be removed before the O-site N-acetylglucosamine glycosylated proteins can be selectively purified by affinity chromatography columns. This deglycosylation process can be achieved by a peptide N-glycosidase F capable of removing both high-mannose-type and complex N-glycosides. However, as mentioned earlier, the effect of peptide N-glycosidase F on plant complex-type glycosides is limited due to the presence of α-1-3 fucose attached to the neighboring N-glycoside center, GlcNAc [ see 25. 3. 2 Section 3. 2 ]. To overcome this disadvantage, it is important to keep the plant material in a high mannose-type glycoside form that is sensitive to PNGaseF. This experimental method was developed in our laboratory for the conversion of N-glycosides from cell cultures of mutants to the Man5GlcNAc2 structure [29] . This experimental method should be applicable to other plant materials to achieve glycosylation of high-mannose-type N-glycosides.
( 1 ) Filter 150 ml of cell culture through a magic filter cloth, then suspend the cell culture in 150 ml of 0.5 mol/L NaCl solution and stir at 4°C for 30 min to dissolve proteins bound to the cell wall by ionic bonds (see Note 16 and Note 17).
( 2 ) Refilter the cell culture through a magic filter cloth. Discard the supernatant and grind the cell culture in 30 ml of 20 mmol/L Tris-HCl, pH 7.8 ( WGA buffer) containing 150 mmol/L KCl, 2 mmol/L CaCl2, 10 mmol/L MgCl2, 1 mmol/L DTT, and protease inhibitors.
( 3 ) The extract was centrifuged at 5500 g for 15 min at 4°C, followed by 25000 g for 30 min, and the insoluble material was discarded and dispensed into 5 ml tubes.
( 4 ) Concentrate the 5 ml fraction to 0.5 ml using a Centricon centrifugal ultrafiltration tube (Amicon Bioseparation YM-10).
( 5 ) After concentration, SDS is added to the sample to make it contain 0.1% SDS, and the glycoproteins are denatured by heating at 100°C for 5 minutes. NonidetP40 is added to give a final concentration of 0.5% (see Note 12). After denaturation, add PNGaseF ( 5 U ) and incubate for 24 h at 37°C with stirring for protein deglycosylation. In this step, only the N-glycosides are eliminated.
( 6 ) Dilute the deglycosylated sample 20 times with WGA buffer, mix it with 100 μl of WGA-agarose, and rotate and oscillate at 4°C for 4 h. Remove unbound proteins from the column by rinsing the resin with 20 times the column volume of WGA buffer.
( 7 ) To elute proteins bound to the column, mix the resin with 5-10 times the volume of WGA buffer containing 0.5 mol/L GlcNAc at 4°C for 30 min (see Note 18). Collect the eluate and repeat the same elution process once. The two eluates are combined and this glycoprotein fraction contains only the O-position N-acetylglucosamine glycosylated protein.
( 8 ) Glycoproteins are precipitated by adding trichloroacetic acid to a final concentration of 12.5%, overnight at 4°C. The proteins are precipitated by centrifugation at 10,000 g for 15 min, the protein precipitate is washed three times with acetone/water [ 9:1 (V/V) ] solution, and the protein precipitate is suspended in an appropriate buffer for 1D electrophoresis.
( 9 ) Separate the O-position N-acetylglucosamine glycosylated protein by 1D SDS-PAGE gel electrophoresis. After staining with Coomassie Brilliant Blue, all visible bands in the gel were collected and digested by trypsin, and the proteins were identified by MALDI-TOF or LCMS/MS.
3.5 Conclusion and outlook
The application of the experimental methods listed in this chapter allows obtaining the function of the glycosides of a plant glycoprotein and their constituent units, the way they are connected. In fact, the same oligosaccharide sequence attached to different glycoproteins, or appearing at different locations in the plant, or at different times in the plant life cycle, may exhibit different functions. Thus, the emerging study of glycoproteomics is opening a new avenue for understanding the roles of N-glycosides and O-glycosides. Bridging the gap in this field will expand our understanding of plant physiology and offer broader perspectives for biochemistry and medicine.