Protocol of Preparation of Glycosylated Protein Samples

An important feature of protein glycosylation is heterogeneity, that is, different sugar chains can be connected to the same site and different sites can also be connected to different sugar chains on the same protein. The heterogeneity of glycosylation makes it difficult to obtain ideal results for the separation and analysis of glycoproteins. At present, the main research strategy for glycosylated proteins is to separate and enrich glycoproteins/glycopeptides, eliminate the heterogeneity of glycosylation and its impact on mass spectrometry, and label glycosylation sites with quality, so as to realize the identification of large-scale high-throughput glycoproteins and glycosylation sites. At present, the commonly used separation and enrichment technologies for glycoproteins include lectin affinity technology, hydrazine chemical enrichment method, hydrophilic interaction chromatography β-eliminate Michael addition reaction, etc.

Experimental Principle

Complex samples (e.g., membrane proteins, serum, etc.) often contain a wide variety of proteins and the concentrations vary widely between proteins, with the majority of proteins in the sample at low concentrations, except for a very small number of proteins that are more abundant. In glycoprotein research, the large number and high abundance of non-glycoproteins can greatly affect the analysis of glycoproteins, and therefore they need to be enriched before glycosylated proteins can be analyzed. The enrichment efficiency and specificity of the enrichment method play a key role in the results of the subsequent steps. The main current methods for glycoprotein/glycopeptide enrichment include chromatography and chemical modification methods. The main lectins for glycosylated protein enrichment are shown in Table 1-3-4.

Table 1-3-4 Major Lectins for Glycosylated Protein Enrichment

Type		Types of isolated sugar chains
Galactocyte-binding lectin (galectin)	High mannose type or complex type of N-linked glycan chains	N-linked and O-linked sugar chains containing N-acetyl lactosamine (LacNAc).
Concomitant cuttle bean agglutinin A (conA)		Sugar chains containing N-link.
Peanut agglutinin (PNA)	T-antigen of O-linked glycan chains (Galβ1-3GalNAc)	O-linked sugar chain containing T-antigen structure (Galαl -3GalNAc).
Aleuria aurantia Agglutinin (AAL)		Sugar chain of L-fucose.

1. Chromatography Method

(1) Lectin affinity chromatography

Principle. Lectin can selectively combine with specific sugar chains to specifically enrich certain sugar chains or glycoproteins. It is the most widely used separation and enrichment technology in glycoproteomics.

Advantage. The technology is relatively mature, easy to operate, and has good repeatability. Various types of glycoproteins can be classified and enriched.

Disadvantages. The cost of lectin is relatively high, and one lectin cannot enrich all types of glycoproteins in cells. It is necessary to combine a variety of different lectins to obtain various glycoproteins in a sample.

(2) Boronate affinity chromatography

Principle. Boric acid can react reversibly with any compound containing 1,2-cis dihydroxy group. It can covalently combine under alkaline conditions and decompose under acidic conditions. Boric acid can be fixed on the medium to separate and enrich glycoproteins/glycopeptides in protein samples. The "Boric acid lectin affinity chromatography (BLAC)" was prepared by mixing lectin and boric acid affinity column. After combining with glycoprotein, glycoprotein was enriched by one-time or step-by-step elution according to actual needs.

(3) Hydrophilic interaction chromatography

Principle. Sugar chains have strong hydrophilicity. Comparatively speaking, non-glycosylated proteins or peptides have stronger hydrophobicity. Many hydrophilic chromatography media, such as cellose and sepharose, can be used to enrich glycoproteins/glycopeptides.

Advantage. It is easy to operate and can enrich various types of glycopeptides non-selectively.

Disadvantages. This method belongs to non-specific adsorption, and cannot effectively distinguish different types of glycopeptides. A small number of non-glycoproteins that have not been removed will have a certain impact on the subsequent study of glycosylation sites.

(4) Molecular sieve enrichment method

Principle. The molecular weight of glycopeptides after trypsin digestion was significantly higher than that of non-glycosylated peptides. Good results can be obtained by non-selective separation of glycopeptides and non-glycosylated peptides using molecular sieves.

Advantage. It is easy to operate and can enrich various types of glycopeptides non-selectively.

Disadvantages. The efficiency of enzymatic digestion is required to be high. If the digestion is incomplete, the high molecular weight fragments that are not completely digested will seriously affect the enrichment of glycopeptides, which belongs to non-specific adsorption, limiting the subsequent study of glycosylation sites.

(5) Antibody affinity chromatography

Principle. The specific glycoprotein was enriched by antigen-antibody reaction.

Advantage. It is able to specifically study proteins or peptides containing specific sugar chain structures.

Disadvantages. It is expensive, and non-specific adsorption can cause false positive results.

2. Chemical Modification Method

(1) Hydrazine chemical enrichment method

Principle. By modifying oxidized sugars with hydrazide reagents, glycosylated proteins/glycopeptides can be selectively separated and enriched, thus reducing the complexity of the samples to be studied.

Advantage. Different types of glycoproteins/glycopeptides can be enriched indiscriminately at one time, and specific glycoproteins/glycopeptides can be collected by eluting in turn with different methods.

Disadvantages. It belongs to chemical reaction, with many steps and difficult conditions to control.

(2) β-Elimination of Michaelis addition reaction

Adopt β-Elimination of Michaelis addition reaction can selectively enrich the target glycoprotein/glycopeptide by attaching a group with strong reactivity, such as an alkyl group, to the modified site.

Experimental Preparation

1. Main Instruments and Equipment

High-speed centrifuge, ultracentrifuge, ultrasonic crusher, vertical shaker, probe micro-ultrasonic crusher, syringe, magnetic separator.

Qproteome Total Glycoprotein Kit, G-25 demineralization column^*1, hydrazine magnetic particles.

2. Experimental Materials

Protein sample of interest, usually a mixture of multiple proteins that have not been isolated.

3. Main reagents

(1) Lysis Solution

Lysis solution A (applicable to the extraction of water-soluble protein)
40 mmol/L	Tris-base (pH 9. 5)
Lysis solution B (classic formula)
8 mol/L	Urea
4%	CHAPS
40 mmol/L	Tris-base
1 %	DTT
1 %	Protease inhibitor
Lysis solution based on classical formula (C - E)
Lysis solution C
7 mol/L	Urea
2 mol/L	Thiourea
2 % - 4 %	CHAPS
40 mmol/L	Tris-base
1 %	DTT
2 %	Pharmalyte (pH 3 - 10)
1%	Protease inhibitor
Lysis solution D
9.5 mol/L	Urea
2 %	CHAPS
1 %	Tris-base
0.8 %	Pharmalyte (pH 3 - 10)
5 mmol/L	Protease inhibitor
Lysis solution E
9.5 mol/L	Urea
2 % - 4 %	CHAPS
1 %	DTT
2 %	Pharmalyte (pH 3 - 10)
5 mmol/L	Protease inhibitor
Lysis solution F (applicable to membrane protein extraction)
5 mol/L	Urea
2 mol/L	Thiourea
2 %	SB 3-10
2 %	CHAPS
1 %	DTT
0.5 %	CA
5 mmol/L	Protease inhibitor
Lysis solution G (applicable to the extraction of insoluble precipitated protein)
1%	SDS
0. 375 mol/L	Tris-HCl (pH 8.8)
50 mmol/L	DTT
25% (volume)	Glycerol
Lysis solution H（applicable to the PF-2D）
7.5 mol/L	Urea
2.5 mol/L	Thiourea
12.5 %	Glycerol
62.5 mmol/L	Tris-HCl
2. 5 %	n-Octyl-β-D-Glucopyranoside
6.25 mmol/L	Tris (2-carboxyethyl) phosphine TCEP
1.25 mmol/L	Protease inhibitor

(2) Tris, TBS, PBS, HEPES buffer, acetone

(3) Binding buffer, protease inhibitor, descaler, elution buffer according to QproteomeTM Glycoprotein Fractionation Handbook, QIAGEN

(4) Epoxide magnetic particles

(5) Coupling buffer (pH 8.5)：

100 mmol/L	NaAC
150 mmol/L	NaCl

(6) Washing buffer

8 mol/L	Urea
0. 4 mol/L	NH4HCO3 (pH 8.3)

(7) Other reagents

1 mol/L NH₄HCO₃ (pH 7. 8), iodoacetalamine, trypsin, 2 mol/L NaCl, anhydrous ethanol, glycopeptidase (PNGaseF).

(8) Protease inhibitors

In the process of tissue cell fragmentation, the addition of diisopropyl fluorophosphate (DFP) can inhibit or slow down autolysis. The addition of iodoacetic acid can inhibit the activity of proteolytic enzymes that require sulfhydryl groups in active centers. The activity of proteolytic enzyme can also be eliminated by adding benzene sulfonyl fluoride (PMSF), which can be added according to the actual conditions in the experiment (Table 1-3-5). Use protease inhibitor complex tablets (Roche molecular Biochernicals), 5 mg/mL peptide inhibitor^*2 prepared with 10 g/mL of DMSO as solvent.

Table 1-3-5 Broad-spectrum Protease Inhibitor Mixture

Component	Final concentration
PMSF	35 μg/mL (1 mmol/L)
EDTA	0.3 mg/mL (1 mmol/L)
Pepstatin	0.7 μg/mL
Leupeptin	0.5 μg/mL

Experimental Methods

Experiment 1. Enrichment of Total Cellular Glycoproteins by Lectin Assay (Referring to Qproteome Total Glycoprotein Kit)

(1) Cells were collected by scraping and centrifuged at 450g for 5 min washing the precipitate twice with HEPES buffer or TBS and the cells were placed on ice^*3.
(2) During centrifugation, 3 mL of binding buffer [containing 30 μL of protease inhibitor (100×) and 300 μL of descaler] was added to the centrifuge column.
(3) Gently aspirate the suspended cell precipitate by adding binding buffer [containing 30 μL protease inhibitor (100×) and 300 μL descaler] in an amount of approximately 1 mL of binding buffer for 1 × 10⁷ cells.
(4) Incubate at 4°C for 15 min with vortex shaking every 5 min.
(5) Slowly aspirate the cell precipitate 10 times with a syringe^*4 and agitate the cells well.
(6) Centrifuge the cell lysate at 10,000g at 4°C for 20 min.
(7) Prepare the centrifuge tube during centrifugation by loosening the cap 1/4 of the tube, removing the top cap, placing the centrifuge column into an empty 2mL collection tube, and centrifuging the column at 500r/min for 2min.
(8) Discard the flow-through solution, add 500 μL of binding buffer to the centrifuge column, and centrifuge the column at 500 r/min for 2 min.
(9) Discard the flow-through solution, add 500 μL of the post-centrifugation lysate supernatant in (6) to the centrifugation column.
(10) Incubate for 1 min and centrifuge at 500 r/min for 2 min^*5.
(11) Add another 500 μL of the post-centrifugation lysate supernatant from (6) to the centrifuge column. Repeat (10) and (11) once.
(12) Add 750 μL of binding buffer [containing 30 μL of protease inhibitor (100×) and 300 μL of descaler] to wash the centrifugation column, centrifuge at 500 r/min for 2 min, discard the flow-through solution, and repeat this step once.
(13) Place the centrifuge column in a new centrifuge tube, add 100 μL elution buffer [containing 3 μL protease inhibitor (100×) and 30 μL descaler] and incubate for 1 min, centrifuge for 2 min at 500 r/min.
(14) Repeat the previous step 3 times.
(15) The eluates from the 4 times were combined, and the concentration could be determined by Lowry's method.

Experiment 2. Enrichment of Total Glycoproteins of Tissue by Lectin Assay (Referring to Qproteome Total Glycoprotein Kit)

(1) 3 mL of binding buffer [containing 30 μL of protease inhibitor (100×) and 300 μL of detergent] was added to the centrifuge column.
(2) Add 1mL of binding buffer [containing 30μL of protease inhibitor (100×) and 300μL of descaler] to the tissue and homogenize for 30s at the lowest speed using a TissueRuptor (handheld homogenizer).
(3) Incubate for 15 min at 4°C with vortex shaking every 5 min.
(4) Cell lysate was centrifuged at 1000g at 4°C for 20min.
(5) Prepare the centrifuge tube during centrifugation by loosening the cap by 1/4, removing the top cap, placing the column in an empty 2 mL collection tube, centrifuging the column at 500 r/min for 2 min. discard the flow-through solution, adding 500 μL of binding buffer to the column, and centrifuging at 500 r/min for 2 min. discard the flow-through solution.
(6) Add 500 μL of the post-centrifugation lysate supernatant from (5) to the centrifugation column. Incubate for 1 min and centrifuge at 500 r/min for 2 min. If unglycosylated proteins are to be analyzed, collect the flow-through fraction.
(7) Repeat (6) once.
(8) Add 750 μL of binding buffer [containing 30 μL of protease inhibitor (100×) and 300 μL of detergent] and wash the centrifuge column, centrifuge at 500 r/min for 2 min, discard the flow-through solution, and repeat this step once.
(9) Place the centrifuge column into a new centrifuge tube, add 100 μL elution buffer [containing 3 μL protease inhibitor (100×) and 30 μL descaler] and incubate for 1 min, centrifuge at 500 r/min for 2 min.
(10) Repeat the previous step 3 times.
(11) The four eluates were combined and the concentration could be determined by Lowry's method.
(12) Add 4 times the volume of acetone to the protein sample and incubate on ice for 15 min^*6.
(13) Centrifuge at 4°C for 10 min at 12000 r/min.
(14) Discard the supernatant and air dry the precipitate^*7.
(15) A suitable solvent can be selected to dissolve the precipitate according to subsequent experiments.

Experiment 3. Separation and Enrichment of Glycoproteins by Hydrazine Magnetic Particles^*8

(1) Oxidation of glycoproteins by periodate: A certain amount of protein sample was dissolved in coupling buffer (100 mmol/L NaAC, 150 mmol/L NaCl, pH 8.5), and NaOH solution was added to its final concentration of 15 mmol/L. The reaction was carried out at room temperature and protected from light for 1h.
(2) Removal of unreacted NaIO₄. The oxidized protein solution was added to the G-25 desalting column pretreated with coupling buffer, and when all the protein solution entered the desalting column, coupling buffer was added to the column, and the effluent solution was picked up by a 0.5 mL centrifuge tube, and each tube with absorbance value greater than 0.1 at 280 nm was combined to obtain the oxidized glycoprotein solution without IO₄-.
(3) Coupling/extraction. Mix the oxidized glycoprotein solution with the hydrazine pellets pretreated with coupling buffer, and shake the pellets for more than 6 h at room temperature on a shaker^*9.
(4) Washing. The coupled magnetic pellets were magnetically separated and the supernatant was removed. The pellets were washed 3-6 times with washing solution (8 mol/L urea, 0.4 mol/ NH₄HCO₃, pH 8.3) to remove the uncovalently bound proteins, and the glycoproteins were purified and enriched on the magnetic pellets.

Experiment 4. Separation and Enrichment of Glycopeptide by Hydrazine Magnetic Particles

(1) Reduction/oxidation. The glycoproteins on the magnetic particles were reduced with 5 mmol/L TCEP (dissolved in 0.1 mmol/L NH4HCO3 solution, pH 7.8) at room temperature for 30 min.
(2) The reaction was continued at room temperature for 30 min with the addition of iodoacetophenolamine to a final concentration of 10 mmol/L.
(3) Trypsin digestion. After oxidation, the pellets were magnetically separated, the supernatant was removed, and the pellets were resuspended with NH4HCO3. pH 7.8. The pellets were added with trypsin to a final concentration of 20 mmol/L, and the enzymatic digestion was shaken at 37°C for 4 h. The same amount of enzyme was added and the enzymatic digestion was continued for 18 h.
(4) After enzymatic digestion, magnetic separation was performed, and the supernatant was removed. The magnetic pellets were washed 3-5 times with 2 mol/L NaCl, anhydrous ethanol, respectively.
(5) Release of glycopeptides. After trypsin digestion, the magnetic pellets were pre-washed with 0.1 mol/L NH4HCO3. solution for 2 times and then resuspended with NH4HCO3 solution. Add 0.5 μL of PNGaseF, shake the enzymatic digestion for 4 h at 37°C, add the same amount of enzyme, and continue the reaction overnight. Separate magnetically and collect the supernatant, which is the deglycosylated peptide.

Note

*1 Centrifuge columns should be used with attention to their maximum volume that can be accommodated and the maximum amount of protein bound.
*2 Must be freshly prepared on the same day.
*3 Do not wash with phosphate buffer in this step to prevent it from interfering with the binding of glycoproteins to the lectin resin.
*4 Be equipped with a blunt-ended needle to prevent damage to the cell structure during aspiration.
*5 For analysis of unglycosylated proteins, collect the flow-through fraction.
*6 As the amount of enriched protein is still relatively small, acetone precipitation can be used for concentration and desalting.
*7 Be careful not to over-dry (it can lead to insoluble precipitation).
*8 Refer to Sun Shisheng. Study of a new method for enrichment of glycoproteins/glycopeptides by magnetic particles. Xi'an: Northwestern University, 2008.
*9 This step should be done for sufficient time to ensure that the oxidized glycoproteins react fully with the magnetic particles.