CN104236983A - Method for removing ionic liquid containing long alkyl chain in solution sample - Google Patents

Abstract

The invention relates to a method for removing ionic liquids containing long alkyl chains. The removal method comprises the following steps: a) adjusting the solution containing ionic liquids to be alkaline (pH ≥ 8); b) using cation exchange materials as adsorbents , removing the ionic liquid containing long alkyl chains; c) collecting the solution after removing the ionic liquid. This method can well remove ionic liquids containing long alkyl chains in the solution, and has the advantages of simplicity, efficiency and speed, and has broad application prospects in proteomics, polymer chemistry and liposome metabolomics .

Description

Removal method of ionic liquid containing long alkyl chain in solution sample

technical field

The invention relates to a method for removing an ionic liquid containing a long alkyl chain and its application, which can realize the effective removal of an ionic liquid containing a long alkyl chain in a solution, and has the advantages of being simple, efficient and fast, and the method can be widely used It is used in the research of proteomics, polymer chemistry and liposome metabolomics.

Background technique

Membrane proteins play an important role in the exchange of substances inside and outside the cell, cell recognition and immune response, signal transduction and regulation, and energy transfer. 1/3 of the proteins in eukaryotes are integrated on the membrane. In addition, membrane proteins also play a very important role in drug research, and about 70% of known and researched drug targets are membrane proteins. However, due to the strong hydrophobicity of membrane proteins, their solubility and enzymatic hydrolysis efficiency are poor, making analysis difficult. Therefore, choosing a solvent with high solubility for membrane proteins is a prerequisite for membrane protein research.

In recent years, ionic liquids have attracted more and more attention as a promising green solvent. Ionic liquids are composed entirely of anions and cations, and usually behave as liquids at room temperature or salts with a melting point of less than 100°C. Ionic liquids have the advantages of good solvent resistance, non-volatility, thermal stability, wide liquid range, and anion and cation designability, and have been widely used in many fields such as chemical synthesis, electrochemistry, extraction and separation, and material preparation. Due to their good solubility, ionic liquids have become new solvents for the dissolution of membrane proteins in recent years (Sun, L.L.; Tao, D.Y.; Han, B.; Ma, J.F.; Zhu, G.J.; Liang, Z.; Shan, Y.C.; Zhang , L.H.; Zhang, Y.K. Anal. Bioanal. Chem. 2011, 399, 3387-3397.).

"Bottom-up" technology is currently the most commonly used technology in proteomics analysis and identification. This technology first enzymatically hydrolyzes proteins into peptides, and then separates them through one-dimensional or multi-dimensional separation technology and identifies them by online mass spectrometry. In the analysis of membrane proteins, for the solubilization of membrane proteins, the addition of solubilizers such as ionic liquids will seriously affect the mass spectrometry signal of protein analysis. Therefore, before mass spectrometry analysis, it is necessary to remove solubilizers such as ionic liquids from protein samples to obtain ideal mass spectrometry signals.

Contents of the invention

In order to solve the above problems, the purpose of the present invention is to develop a method for removing ionic liquids containing long alkyl chains, by which the ionic liquids in the solution can be removed efficiently, quickly and easily, thereby being compatible with the subsequent analysis process , which will not adversely affect the subsequent analysis.

To achieve the above object, the technical solution adopted in the present invention is:

The method for removing the ionic liquid containing long alkyl chains in the solution sample is to adjust the sample solution of the ionic liquid containing long alkyl chains to an alkaline environment with pH ≥ 8 with an alkaline solution; then use a cation exchange material as an adsorbent, The ionic liquid containing long alkyl chains is removed, and finally the adsorbent is separated from the solution, and the solution is collected.

The ionic liquid containing a long alkyl chain can be: the cation part is an alkyl chain part, which is an imidazole, pyridine, quaternary ammonium, or quaternary phosphonium cation containing 6 carbons or more than 7 carbons; the anion part is Cl ^- , Br ^- , I ^- , NO ₃ ^- , ClO ₄ ^- , AlCl ₄ ^- , BF ₄ ^- , PF ₄ ^- , CF ₃ COO ^- , CF ₃ SO ₃ ^- , (CF ₃ SO ₂ ) ₂ N ^- or SbF ₆ ^- .

The sample solution of the ionic liquid containing long alkyl chains is adjusted to an alkaline environment with a pH of 8-14 with an alkaline solution.

The sample solution of the ionic liquid containing long alkyl chains can be: cell extract, cytoplasm extract, plasma extract, protein solution, polypeptide solution, lipid solution or electronegative polymer solution.

The alkaline solution used to adjust the pH to an alkaline environment is ammonium bicarbonate buffered saline solution with a pH of 9-14, phosphate buffered saline solution with a pH of 9-14, tris buffered saline with a pH of 9-14 Salt solution, ammonia, sodium carbonate solution, or sodium hydroxide solution.

The cation exchange material is a silica gel or a polymeric matrix material (e.g. polyacrylate matrix, polystyrene matrix, polyacrylamide matrix) containing sulfonic and/or phosphoric acid groups; the material can be granular or overall material.

The manner of contacting and separating the adsorbent and the sample solution may be:

The adsorbent is immobilized on the solid phase carrier or filled in a hollow container, and the sample solution containing the ionic liquid passes through the solid phase carrier or the hollow container, and is directly collected after contacting with the adsorbent;

Or after the adsorbent and the sample solution are mixed and contacted, the adsorbent is separated from the solution containing the ionic liquid by centrifugal precipitation;

Or wrap the adsorbent on the magnetic particles, mix and contact the adsorbent with the sample solution, and separate it from the sample solution containing the ionic liquid by magnetic force.

The solid phase carrier is: the surface area is 1cm ² -10m ² , the shape is one or more of cuboid, cube, cone or cylinder, and the material is polymer material, specifically silica gel, cellulose membrane, polyethersulfone membrane , resins, dextran gels, agarose gels, and magnetic composites.

The hollow container is: a stainless steel tube with a diameter of 50 μm-5 cm in the radial cross-section of the internal cavity, a pipette tip of 20-1000 μl, a solid phase extraction (SPE) tube of 1-200ml, a syringe needle tube of 1-200ml, and an inner diameter of 50-500μm Capillary or syringe filter chamber.

The sample solution is a mixture of one or two or more of protein samples, liposome samples and macromolecular samples, and the method can be used in the analysis of protein samples, liposome samples and macromolecular samples.

specific:

1. With the sample solution of the ionic liquid containing the long alkyl chain, the pH is 9-14 ammonium bicarbonate buffered saline solution, phosphate buffered saline solution, Tris hydroxymethylaminomethane buffered saline solution, ammonia water, sodium carbonate solution, or The alkaline solution of sodium hydroxide solution is adjusted to an alkaline environment with pH>8;

2. Using silica gel or polymer matrix cation exchange material containing sulfonic acid groups and/or phosphoric acid groups as the adsorbent to absorb the ionic liquid containing long alkyl chains in the solution. If the adsorbent is immobilized on a solid-phase carrier or filled in a hollow container, the sample solution containing a long alkyl chain ionic liquid can be directly passed through the adsorbent at a flow rate of 10-1 000 μL; if the adsorbent is not immobilized If the particles are liquefied, add the adsorbent directly into the solution containing the long alkyl chain ionic liquid, and shake for 3-10 minutes.

3. If the adsorbent is immobilized on a solid-phase carrier or filled in a hollow container, the effluent is collected directly, which is the solution after removing the ionic liquid; if the adsorbent is an unimmobilized particle, it is centrifuged (100-30000g) separate the adsorbent and the solution, and collect the solution part, which is the solution after removing the ionic liquid.

The present invention has the following advantages:

1. High removal efficiency. In the alkaline range of pH>8, most proteins or peptides become electronegative. Since the negative charge is opposite to the positive charge of cations containing long alkyl chains in ionic liquids, strong cation exchange materials are used, which contain long The ionic liquid of the alkyl chain is retained, but not the protein or peptide, thereby separating the ionic liquid and the protein/peptide.

2. The operation is convenient and fast. The strong cation exchange material only needs to be in full contact with the ionic liquid containing long alkyl chains to separate the two. Subsequently, the solution after removing the ionic liquid can be recovered by means of centrifugation or magnetic separation. If the strong cation exchange material is packed in the column tube or immobilized, the solution containing the ionic liquid is passed through the cation exchange material, and the effluent is collected to be the solution after the ionic liquid is removed.

Description of drawings

Figure 1 is the MALDI-TOF mass spectrum of the BSA hydrolysis product. Figure 1. Using a strong cation exchange material as an adsorbent (A) filled it in a trapping column (B) put it directly into the solution and removed the mass spectrum of the BSA enzymatic liquid; (C) without ions Mass spectrum of liquid BSA hydrolyzate; (D) Mass spectrum of BSA hydrolyzate containing ionic liquid.

Detailed ways

Example 1

1. Bovine serum albumin (BSA) sample pretreatment: Dissolve 1 mg BSA in 1 mL of 4% (4 g/100 mL) 1-dodecyl-3-methylimidazole chloride (C12Im-Cl) in 50 mM ammonium bicarbonate solution ( In ABC), after thermal denaturation at 90°C for 10min, 10mM dithiothreitol (DTT) solution was added, reacted at 56°C for 60min for protein reduction treatment, then passed into 30mM iodoacetamide (IAA) solution, and kept in room temperature After 30 minutes of light reaction, the protein was alkylated. Finally, according to protein/trypsin: 25/1, 40 μg of trypsin (dissolved in 50 mM ammonium bicarbonate solution, pH 8.0) was added, and the enzyme was hydrolyzed at 37°C for 12 hours. Finally, 10 μl of formic acid was added to acidify and terminate the enzymatic hydrolysis.

2. Removal of ionic liquids containing long alkyl chains: strong cation exchange materials (Tosohatsu, Japan, TSK-GEL SP-5PW, 10 μm, 1000 ) is filled in the polished stainless steel trap column tube (inner diameter 4.6cm, length 1cm). To the enzymatic hydrolyzate (100 μL, 50 mM ABC dissolved in 4% C12Im-Cl) with a molar concentration of 1 mg/mL BSA was added 400 μL of 1 mM Tris-HCl buffered saline (Tris-HCl, pH 9.5). Subsequently, the above-mentioned 0.2mg/mL BSA enzymatic hydrolysis product was used as a carrier liquid with 1mM Tris-HCl, pH9.5, and was driven by a pump at a flow rate of 1mL/min, and passed through a strong cation exchange trapping column to collect the effluent. That is, the solution after removing the ionic liquid.

3. MALDI-TOF analysis: After lyophilizing the collected BSA hydrolyzate solution after removing the ionic liquid, redissolve it in 500 μL of 0.1% (volume fraction) trifluoroacetic acid aqueous solution, that is, 0.2 mg/mL BSA hydrolyzate. Take 2-cyano-4-hydroxycinnamic acid (7 mg/mL, dissolved in 60% acetonitrile aqueous solution containing 0.1% (volume fraction) trifluoroacetic acid) as matrix, mix according to volume ratio 1:1, apply 2 μL, and naturally After air drying, MALDI-TOF detection. The mass spectrum is shown in Figure 1 (A), and the identification results are shown in Table 1.

Example 2

1. Bovine serum albumin (BSA) sample pretreatment: Dissolve molar concentration of 1 mg BSA in 1 mL of 4% (4 g/100 mL) 1-dodecyl-3-methylimidazole chloride (C12Im-Cl) in 50 mM bicarbonate In ammonium solution (ABC), after thermal denaturation at 90°C for 10min, add 10mM dithiothreitol (DTT) solution, react at 56°C for 60min for protein reduction treatment, and pass through 30mM iodoacetamide (IAA) solution, After reacting in the dark for 30 minutes at room temperature, the protein was alkylated. Finally, according to protein/trypsin: 25/1, add 40 μg of trypsin (dissolved in 50 mM ammonium bicarbonate solution, pH 8.0), and enzymatically hydrolyze at 37°C for 12 hours. Finally, 10 μl of formic acid was added to acidify and terminate the enzymatic hydrolysis.

2. Removal of ionic liquids containing long alkyl chains: Add 400 μL of 1 mM tris-hydrochloric acid buffered saline (Tris- HCl, pH9.5). Subsequently, 2 mg of strong cation exchange material (Tosohatsu, TSK-GEL SP-5PW, 10 μm, 1000 ), after shaking for 3 minutes, centrifuge at 14000g for 5 minutes. Take the supernatant, which is the solution after removing the ionic liquid.

3. MALDI-TOF analysis: After lyophilizing the collected BSA hydrolyzate solution after removing the ionic liquid, redissolve it in 500 μL of 0.1% (volume fraction) trifluoroacetic acid aqueous solution, that is, 0.2 mg/mL BSA hydrolyzate. Take 2-cyano-4-hydroxycinnamic acid (7 mg/mL, dissolved in 60% acetonitrile aqueous solution containing 0.1% (volume fraction) trifluoroacetic acid) as matrix, mix according to volume ratio 1:1, apply 2 μL, and naturally After air drying, MALDI-TOF detection. The mass spectrum is shown in Figure 1(B), and the identification results are shown in Table 1.

Control group 1

Dissolve BSA in a solvent that does not contain ionic liquids with long alkyl chains for sample pretreatment: dissolve 1 mg of BSA in 1 mL of 50 mM ammonium bicarbonate solution (ABC), heat denaturation at 90 ° C for 10 min, and then add 10 mM dithiothreitol (DTT) solution, reacted at 56°C for 60 minutes for protein reduction treatment, then passed through 30 mM iodoacetamide (IAA) solution, reacted at room temperature for 30 minutes in the dark, and then carried out protein alkylation treatment. Finally, according to protein/trypsin: 25/1, 40 μg of trypsin (dissolved in 50 mM ammonium bicarbonate solution, pH 8.0) was added, and the enzyme was hydrolyzed at 37°C for 12 hours. Finally, 10 μl of formic acid was added to acidify and terminate the enzymatic hydrolysis. Dilute the BSA enzymatic hydrolysis product to 0.2 mg/mL, use 2-cyano-4-hydroxycinnamic acid (7 mg/mL, dissolved in 60% acetonitrile aqueous solution containing 0.1% (volume fraction) trifluoroacetic acid) as the matrix, press Mix at a volume ratio of 1:1, apply 2 μL of the sample, and let it dry naturally for MALDI-TOF detection. The mass spectrum is shown in Figure 1(C), and the identification results are shown in Table 1.

Control group 2

Use the BSA hydrolyzate solution containing long alkyl chain ionic liquid as the control group: dissolve 1mg BSA in 1mL 4% (4g/100mL) chloride 1-dodecyl-3-methylimidazole (C12Im-Cl) In the 50mM ammonium bicarbonate solution (ABC), after heat denaturation at 90°C for 10min, add 10mM dithiothreitol (DTT) solution, react at 56°C for 60min for protein reduction treatment, pass through 30mM iodoacetamide ( IAA) solution, reacted in the dark for 30min at room temperature, and then carried out the alkylation treatment of the protein. Finally, according to protein/trypsin: 25/1, 40 μg of trypsin (dissolved in 50 mM ammonium bicarbonate solution, pH 8.0) was added, and the enzyme was hydrolyzed at 37°C for 12 hours. Finally, 10 μl of formic acid was added to acidify and terminate the enzymatic hydrolysis. Dilute the BSA enzymatic hydrolysis product to 0.2 mg/mL, use 2-cyano-4-hydroxycinnamic acid (7 mg/mL, dissolved in 60% acetonitrile aqueous solution containing 0.1% (volume fraction) trifluoroacetic acid) as the matrix, press Mix at a volume ratio of 1:1, apply 2 μL of the sample, and let it dry naturally for MALDI-TOF detection. The mass spectrum is shown in Figure 1(D), and the identification results are shown in Table 1.

Table 1. Identification results of BSA hydrolysis products

Number of identified peptides Identified sequence coverage Figure 1(A) 32 55% Figure 1(B) 30 51% Figure 1(C) 30 53% Figure 1(D) / /

It can be seen from Table 1 and Fig. 1 that by the method of the invention, the ionic liquid containing long alkyl chains can be efficiently removed from the solution, thus not affecting the detection of mass spectrometry.

Claims (9)

1. the removal method of the ionic liquid containing long alkyl chain in the solution sample, it is characterized in that: the sample solution of the ionic liquid containing long alkyl chain is adjusted to the alkaline environment of pH ≥ 8 with alkaline solution; Then adopt cationic The exchange material is used as an adsorbent to remove the ionic liquid containing long alkyl chains, and finally the adsorbent is separated from the solution to collect the solution. 2. The removal method according to claim 1, characterized in that: the ionic liquid containing long alkyl chains can be: the cationic part is an alkyl chain part, which is imidazoles, pyridines containing 6 carbons or more than 7 carbons Class, quaternary ammonium, or quaternary phosphonium cations; the anion part is Cl ^- , Br ^- , I ^- , NO ₃ ^- , ClO ₄ ^{- , AlCl 4 -} _, ^BF ₄ ^- , PF ₄ ^- , CF ₃ COO ^- , CF ₃ SO ₃ ^- , (CF ₃ SO ₂ ) ₂ N ^- or SbF ₆ ^- . 3. The removal method according to claim 1, characterized in that: the sample solution of the ionic liquid containing long alkyl chains is adjusted to an alkaline environment with a pH of 8-14 with an alkaline solution. 4. The removal method according to claim 1, characterized in that: the sample solution containing the ionic liquid of long alkyl chain can be: cell extract, cytoplasm extract, plasma extract, protein solution, polypeptide solution, lipid Matter solution or electronegative polymer solution. 5. The removal method according to claim 1, characterized in that: the alkaline solution used to adjust pH to alkaline environment is ammonium bicarbonate buffered saline solution with pH of 9-14, phosphate buffered solution with pH of 9-14 Saline solution, tris buffered saline solution with a pH of 9-14, ammonia water, sodium carbonate solution, or sodium hydroxide solution. 6. The removal method according to claim 1, characterized in that: the cation exchange material is silica gel or polymer matrix material (polyacrylate matrix, polystyrene matrix) containing sulfonic acid groups and/or phosphoric acid groups , polyacrylamide matrix); the material can be granular or monolithic. 7. removal method according to claim 1 is characterized in that: the mode that adsorbent and sample solution are contacted and separated can be: The adsorbent is immobilized on the solid phase carrier or filled in a hollow container, and the sample solution containing the ionic liquid passes through the solid phase carrier or the hollow container, and is directly collected after contacting with the adsorbent; Or after the adsorbent and the sample solution are mixed and contacted, the adsorbent is separated from the solution containing the ionic liquid by centrifugal precipitation; Or wrap the adsorbent on the magnetic particles, mix and contact the adsorbent with the sample solution, and separate it from the sample solution containing the ionic liquid by magnetic force. 8. The removal method according to claim 6, characterized in that: The solid phase carrier is: the surface area is 1cm ² -10m ² , the shape is one or more of cuboid, cube, cone or cylinder, and the material is polymer material, specifically silica gel, cellulose membrane, polyethersulfone membrane , resins, dextran gels, agarose gels, and magnetic composites. The hollow container is: a stainless steel tube with a diameter of 50 μm-5 cm in the radial cross-section of the internal cavity, a pipette tip of 20-1000 μl, a solid phase extraction (SPE) tube of 1-200ml, a syringe needle tube of 1-200ml, and an inner diameter of 50-500μm Capillary or syringe filter chamber. 9. The removal method according to claim 1, characterized in that: the sample solution is a mixture of one or more of protein samples, liposome samples and polymer samples, and the method can be used for protein samples, lipid samples, In the analysis of body samples and polymer samples.