WO2014198113A1

WO2014198113A1 - Method for removing from a solution sample ionic liquid containing long alkyl chains

Info

Publication number: WO2014198113A1
Application number: PCT/CN2013/090129
Authority: WO
Inventors: 张丽华; 赵群; 杨开广; 方菲; 张玉奎
Original assignee: 中国科学院大连化学物理研究所
Priority date: 2013-06-14
Filing date: 2013-12-20
Publication date: 2014-12-18
Also published as: CN104236983A

Abstract

A method for removing ionic liquid containing long alkyl chains, comprising the following steps: a) alkalizing a solution containing ionic liquid (pH≥8); b) using a cation exchange material as an adsorbent to remove ionic liquid containing long alkyl chains; c) collecting the solution after removal of ionic liquid. The method can amply remove from a solution sample ionic liquid containing long alkyl chains, has the advantages of simplicity, efficiency, and rapidity, and also has broad prospects for application in proteomics, polymer chemistry, and liposome metabolomics.

Description

Solution sample ¹ 1 'method of removing ionic liquid containing long alkyl chain

Technical field

The invention relates to a method for removing a liquid containing an alkane 3⁄4 chain and an application thereof, and the present invention has the advantages of simple, effective and rapid removal of the liquid from the f-liquid containing the κ alkane 3⁄4 chain. , and 11 This method is widely used in the study of egg n-mass spectrometry, sputum f chemistry and liposome metabolomics.

Background technique

Membrane egg n plays an important role in performing functions such as exogenous substance exchange, cell recognition and immune response, signal transduction and regulation, and energy transfer. The eukaryotic cattle towel 1/3 of the egg η is integrated on the membrane. In addition, Membrane Eggs also plays a significant role in drug research towels, and about 70% of the known and under-explored drug targets are membrane sin. However, the mountain membrane protein is highly hydrophobic, resulting in poor solubility and enzymatic efficiency, and analysis is difficult. Therefore, the choice of a solvent that has a bismuth solubility in the membrane sin is a condition for membrane sin studies.

In recent years, the more attention has been paid to the use of f liquid as a promising green solvent. The isolated body is composed of a mountain yin and yang, and usually exhibits a liquid at room temperature or a salt having a melting point of less than 100 °c. The liquid has good solvent property, small volatilization, heat stability, wide liquid range, and yin and yang design, and is widely used in many fields such as chemical synthesis, electrochemistry, extraction separation, and material preparation. Mountain has a good solubility in recent years, and it has become a new solvent for membrane ίίίΠ dissolution from recent years (Sun, LL; Tao, DY; Han, B.; Ma, JF; Zhu, GJ; Liang, Z.; Shan , Y. C; Zhang, LH; Zhang, YK Anal. Bioanal. Chem. 2011. 599, 3387-3397. ).

The "Bottom-up" technique is the most commonly used technique for the identification of B-pre-egg rig spectroscopy. This technique is to digest the egg mash into peptides, which are separated by one-dimensional or multi-dimensional separation techniques and identified by on-line mass spectrometry. . In the membrane egg enamel analysis towel, which is the solubilization of the sin sin, the added solubilizer such as the f liquid will affect the mass spectrometry signal of the egg η mass analysis. Therefore, before mass spectrometry, it is necessary to remove the solubilizing agent from the Sin sample to obtain a mass spectrometric signal.

Summary of the invention

In order to solve the above i. In the case of J, the invention of B in the invention of the invention is to develop a method for removing the liquid containing the alkane 3⁄4 chain, by which the liquid in the solution can be removed efficiently, quickly and simply, and thus with the continuous analysis process. ? The small continuous analysis caused the impact of small profits.

For the above-mentioned B, the technical solution for the invention of wood is:

The solution of the liquid containing the alkane 3⁄4 chain in the solution sample is removed, and the sample solution containing the alkane 3⁄4 chain from the f liquid is adjusted with a solution of φ. Φ: the environment of pH ≥ 8; then 阳 3⁄4 with cations f3⁄4 The material is used as an adsorbent to remove the liquid containing the alkane 3⁄4 chain, and the adsorbent and the solution are separated most, and the solution is collected.

The liquid leaving the alkane 3⁄4 chain is: the cation moiety is an alkane 3⁄4 chain moiety, which is an imidazole, pyridine, a hand hinge, or a hand squama containing 6 or more carbons. Part is Cr, Br\ Γ, N0 ₃ \ C10 ₄ \ A1C1 BF ₄ \ PF ₄ \ CF ₃ CO(J, CF ₃ SO" (CF ₃ S0 ₂ ) ₂ N- or SbF "

¾ containing alkyl chain K from the sample solution was adjusted with a solution of a liquid sincerity f Φ:. ΡΗ for-environment of _8-14. The liquid-free sample solution containing the alkane 3⁄4 chain is: a cell extract, a cytosol extract, a plasma extract, an egg solution, a polypeptide solution, a liposome solution, or an electronegative polymer solution.

To adjust the pH of the environment, the honest solution is a 9-inch hydrogen carbonate hinge buffer solution, a pH buffer of _9-14 phosphate buffer solution, and a 9- ₁₄ trihydroxymethyl 3⁄43⁄4⁄4 methane buffer salt. Solution, 3⁄4 water, sodium carbonate solution, or hydrogen sodium solution.

The cation-free J" exchange material is a silica gel or a polymer material containing a sulfonic acid group and/or a phosphoric acid group (for example, poly-ene enoic acid, styrene, polystyrene, poly-ene-ene) The amide is a particulate material or a monolithic material. The way in which the adsorbent and the sample solution are contacted and separated is as follows:

Is the adsorbent immobilized on a solid support or filled in a hollow? In the device, the sample solution containing the liquid is passed through

^ Phase carrier or towel empty? ,, and contact with the adsorbent Λ , direct collection;

Or the adsorbent and the sample solution are mixedly contacted, and the adsorbent is separated from the solution containing the ex vivo by centrifugal precipitation;

Or the adsorbent is wrapped on the magnetic particles, and the adsorbent and the sample solution are mixed and contacted with Λ, and separated from the sample solution containing the liquid from the f by magnetic force.

^ Phase carrier is: surface area is lcm ² -K) m ² , the shape is one or more of a square, a cube, a cone or a cylinder, the material is a polymer material, specifically silica gel, fiber * film, poly Ether film, resin, dextran gel, agarose gel and magnetic composite.

Empty? ? The device is: internal cavity diameter ^ small rust steel '笞' with a cross-section diameter of 50 μηι-5 cm, 20-1000 μΐ pipette tip, 1-200 ml | ≤ | phase extraction (SPE)

1-200 ml syringe needle '笞', 50-500 μηι inner diameter capillary '笞' or syringe filter chamber.

The sample solution is one of an egg η sample, a liposome sample, and a sputum sample, or a mixture of sputum samples, 3⁄4 method" - for analysis of egg nucleus samples, liposome samples, and sputum samples .

Specific:

1. A sample solution containing the fc«3⁄43⁄4 chain from the f liquid, using a pH of 9-M bicarbonate hinge buffer solution, phosphate buffered saline solution, trishydroxymethyl 3⁄43⁄4⁄4 methane buffered saline solution, 3⁄4 water, sodium carbonate solution, Or hydrogen 3⁄4 sodium solution in good faith solution. Φ: pH>8 of the honest environment;

2. Using a silica gel or a polymer-containing cationic exchange material containing a sulfonic acid 3⁄4 cluster and/or a phosphate group as an adsorbent, the liquid containing the alkane 3⁄4 chain in the solution is adsorbed. If the adsorbent is carried on the ^ phase carrier or filled in the hollow? For the towel, the sample solution containing the alkane 3⁄4 chain from the f-liquid is directly passed through the adsorbent at a flow rate of 1 (000 μL, that is, if the adsorbent is not determined, the adsorbent is directly added to the alkane-containing 3⁄4 Solution towel with a chain of f liquid, shaking for 3-10 min

3. If the adsorbent is loaded on the ^ phase carrier or filled in the towel? For the towel, the effluent is collected directly, that is, the solution of the effluent is removed; if the sorbent is not granulated by | ≤ |, the adsorbent is separated by centrifugation (100-30 000g) and Solution. Collect the solution part. This is the solution to remove the liquid from the f.

Wood invention has the following advantages:

1. High removal efficiency. The range of satisfies of pH>8 (A), the vast majority of egg yolks or peptides become electronegative, the negative charge of the mountain and the positive charge of the cations containing the alkane 3⁄4 chain from the f liquid, 3⁄4 The material is replaced by a strong cation, and the liquid containing the κ alkane 3⁄4 chain is retained, while the egg η or peptide is smallly retained, thereby separating the f liquid and the si n mass/peptide.

2. Easy to operate and fast to close. The strong cation exchange material only needs to be in full contact with the liquid containing the alkane 3⁄4 chain, that is, to separate the w. With the means of centrifugation or magnetic separation, the solution from the liquid is removed. If the strong cation-exchange material is packed in column A or is determined, the solution containing the liquid from f is passed through the cation-exchange material, and the effluent is collected to remove the solution from the liquid.

DRAWINGS

*1 1 is the MALDI-TOF mass spectrometer of BSA hydrolysate. Winter I 1. Use a strong cation to exchange material for the adsorbent (A) and fill it in the trap column A (B) directly into the solution towel to remove the mass spectrometry product of the BSA from the f liquid. a mass spectrum of a BSA hydrolysate containing a small liquid containing f; (D) a mass spectrum of the BSA hydrolysate containing the liquid from the f.

detailed description

Example 1 1. Bovine Blood Cleanup (BSA) Sample Pretreatment: Dissolve hngBSA in 1 mL of 4% (4 g/100 mL) of 1-Tetra-Alkane 3⁄4-3-methyl 3 imidazole (C ₁₂ Im-Cl) 50 mM hydrogen carbonate hinge solution (ABC) towel, 9 (heat denaturation at 10 min/Ti, adding 10 mM: sulfur «sugar alcohol (DTT) solution, 5 (reaction under TC for 60 min ϋΜϊίίί Π 还 : : : : :: SUTi, pass Into a 30 mM bowl of acetamide (IAA) solution, room '; under the light-proof reaction for 30 min /Tiiit, the egg η alkane 3⁄4 treatment. Most; ΓΪ, according to an / pancreatic an enzyme: 25, add 4ο _μ§ Trypticase η enzyme (dissolved in 50 mM hydrogencarbonate hinge solution, pHS.O), digested for ₁₂ hours at 37 ° C. Most; Γί, acidified with 10 μΐ of formic acid, and finally hydrolyzed.

2. Removal of the liquid containing the alkane 3⁄4 chain: Fill the strong cation exchange material (H Mudong Soda, TSK-GEL SP-5PW, 10 μηι, 1000 Α) on the polished small rust steel trap column'笞' inside (inner diameter 46 cm, K 1cm). ^Molar concentration is imgmL BSA enzymatic hydrolysate (iOO μL, 50 mM ABC dissolved in 4% C12Im-Cl) Add 400 μl^ηιΜtrihydroxymethyl 3⁄43⁄4⁄4 methane-hydrochloric acid Buffered saline solution (Tris-HCl, pH 9.5). With Γί, the above enzymatic hydrolysate of 0.2 mg/mL BSA was used as a carrier liquid with imM Tris-HCl, pH 9.5, pumped at a flow rate of im!Vmin, and the trap column was collected by strong cation exchange. The liquid is the solution for removing the liquid from the f.

3. MALDI-TOF analysis: the collected removal of the liquid from the f; Γί BSA enzymatic solution solution frozen; Ti, fi dissolved in SOO LO.P / o (volume fraction) three 3⁄4 acetic acid aqueous solution, that is, 0.2mgmL BSA enzymatic hydrolysis product. Take

-4-hydroxy 3⁄4 cinnamic acid (7mgmL, dissolved in 60% aqueous solution of acetonitrile containing (U% (volume fraction) tris-3⁄4 acetic acid) is 3⁄4 mass, mixed by volume ratio 1:1, spotting 2 L, cumin wind Ten; Γί, MALDI-TOF detection. Mass spectrometry 1 winter I see 1 winter I 1 (A), the identification results are shown in Table i.

3⁄4 Example 2

1. Nitrogen cleansing nSiri (BSA) sample pretreatment: Dissolve the molar concentration of imgBSA in _1η Λ ₄ % ( _4§ Κ) 0 mL) of 1- _tenx : alkane 3⁄4-3-methyl 3⁄4 imidazole (C ₁₂ Im-Cl) 50 mM hydrogen carbonate hinge solution (ABC) towel, heat-denatured iOmin/Ti at 90 ° C, add 10 mM: sulfur « sugar alcohol (DTT) solution, react at 56 ° C for 60 min ϋί line ίίί 的Also 1 33⁄4 treatment; Γί, access to 30 mM bowl of acetamide (IAA) solution, chamber ';

/ΓίϋΜϊίΐί An alkane treatment. Most; Γί, according to an / pancreatic an enzyme: 25A added, ₄ ο _μ§ of the egg η enzyme (dissolved in 50 mM hydrogencarbonate hinge solution, pH 8.0), enzymatic hydrolysis at 37 ° C for ₁₂ hours. The most fi, add Κ) μΐ formic acid acidification, the final ih enzymatic hydrolysis.

2. Removal of f-liquid containing K alkane 3⁄4 chain: 14 lmgmL BSA enzymatic hydrolysis product (K) 0 L, soluble in 4%

50 mM ABC of C12Im-Cl) 400 μL of ImM Tris-hydroxy- 3⁄43⁄4⁄4 methane-hydrochloric acid buffered saline (Tris-HCl, pH 9.5) was added. ;ί, ^ Add 2 mg of strong cation-free f-exchange material (H Mudong Soda, TSK-GELSP-5PW, ΙΟμηι, 1000 Α) to the above solution, shake for 3 min _/Γί, centrifuge at 14000 g for 5 min. Take the clean, which is to remove the solution from the f liquid.

3. MALDI-TOF analysis: the collected removal of the f liquid; Γί BSA enzymatic solution solution frozen; Ti, fi dissolved in 500 μL 0.1% (volume fraction) three 3⁄4 acetic acid aqueous solution, ie 0.2 mgmL BSA enzymatic hydrolysis product. 4-hydroxy 3⁄4 cinnamic acid (7mgmL, dissolved in 60% aqueous solution of acetonitrile containing (U% (volume fraction) of tri-3⁄4 acetic acid), mixed in a volume ratio of _1:1 , spotted 2 L, cumin Wind ten; Γί, MALDI-TOF detection. Mass spectrometry 1 winter I see 1 winter I 1 (B), the identification results are shown in Table i.

Control group 1 Dissolve BSA with a solvent containing a small amount of K alkane 3⁄4 chain from the f-liquid. Prepare the sample for pretreatment: Dissolve img BSA in imL 50 mM hydrogen carbonate hinge solution (ABC) towel, heat denaturation min / Γί at 90 ° C. Add 10 mM - sulfur « sugar alcohol (DTT) solution, 5 (60 ° TC reaction 60; 1! 1; line egg | "1 quality reduction treatment;:;, into a 30 mM bowl of acetamide (iAA) solution, Room'; lower light-proof reaction; ;1!1; alkane 1⁄4 treatment; i, according to an/tryptic enzyme: 25A, add 40μ _§ of pancreatic egg enzyme (dissolved in 50 mM Hydrogen bicarbonate hinge solution, pHS.O), 3TC enzymatic hydrolysis for 12 hours. Most; Γί, acidification with 10 μΐ of formic acid, final lh enzymatic hydrolysis. BSA digested product diluted 0.2 mg/mL to 2-«3⁄4-4- Hydroxy 3⁄4 cinnamic acid (7mgmL, dissolved in 60% aqueous solution containing OP/((volume fraction) 3⁄4 acetic acid) is 3⁄4 mass, mixed by volume ratio 1:1, spotting 2 L, Π然风十; Γί , MALDI-TOF detection. Mass spectrometry 1 winter I see 1 1 (C), the identification results are shown in Table 1.

Control group 2

The BSA hydrolysate solution containing the alkane 3⁄4 chain from the f liquid was used as the control group: 1 mg of BSA was dissolved in imL 4% (4 g of 100 mL) of chlorinated i -10:alkano 3⁄4-3-methyl 3⁄4 imidazole (C ₁₂ Im- Cl) 50 mM hydrogen carbonate hinge solution (ABC) towel, heat-denatured iOmin/Ti at 90 ° C, add 10 mM -. sulfur « sugar alcohol (DTT) solution, 56 ° (: reaction ₆ 01 ϋΜϊ ίίί Γΐ 1 ί3⁄4 place: SUTi, pass 30 mM bowl of acetamide (IAA) solution, room ';

/ΓίϋΜϊίΐί An alkane treatment. Most; Γί, according to an / pancreatic an enzyme: 25A, adding 4ο _μ§ of the egg η enzyme

(dissolved in 50 mM hydrogencarbonate hinge solution, pH 8.0), digested at 37 ° C for ₁₂ hours. Most fi, add 10 μΐ of formic acid to acidify, and finally lh enzymatically. Dilute the BSA hydrolysate 0.2 mg/mL to 2-3⁄4⁄4-4-hydroxy 3⁄4 cinnamic acid (7mgmL, dissolved in 60% aqueous solution of acetonitrile containing (U% (by volume) 3⁄4 acetic acid). Mixing volume ratio of 1 _:1 , spotting 2 L, Π然风十; Γί, MALDI-TOF detection. Mass spectrometry 1 winter I see 1 winter I 1 (D), the identification results are shown in Table L

Table L Identification results of BSA hydrolysate

From Tables 1 and 1 1 , by the method of the present invention, the liquid containing the alkane 3⁄4 chain is removed from the solution, thereby slightly affecting the detection of the mass spectrum.

Claims

Claim

1. A method for removing a liquid containing an alkane 3⁄4 chain in a solution sample, characterized in that: a sample solution containing an alkane 3⁄4 chain from a liquid is adjusted with a solution of φ. Φ: an environment of pH>8; Λ 3⁄4 Use the cation exchange material as the adsorbent, remove the liquid containing the alkane 3⁄4 chain, and separate the adsorbent and the solution, and collect the solution.

2. The removal method according to claim 1, wherein the de-f liquid containing a K alkane 3⁄4 chain is: the cation moiety is an alkane 3⁄4 chain moiety, and is an imidazole having 6 carbons or more than 7 carbons. Pyridine, .hand hinge, or .hand it is a kind of cation away from the f part of C, Br\ Γ, Ν ( _3⁄4 ·, C10 ₄ \ A1C1 ₄ \ BF ₄ \ PF ₄ \ CF ₃ COO", CF ₃ S0 ₃ \ (CF ₃ S0 ₂ ) ₂ N- or SbF ₆ -.

3. The removing method of claim 1, wherein: alkyl containing K ¾ f chain sample solution from the liquid solution by Cheng modulation Φ:. PH _8-14 for-the environment.

4. The removal method according to claim 1, wherein the sample solution containing the alkane 3⁄4 chain from the f liquid is: a cell extract, a cytosol extract, a plasma extract, a sin solution, a polypeptide solution, a lipid. A solution of the substance or an electronegative polymer solution.

The removal method according to claim 1, wherein: adjusting ρΗ .Φ: Cheng Cheng exemplary environment ¾ solution with ammonium bicarbonate to a pH buffered salt solution is _9-14, the pH is _9-14 phosphate buffered saline, pH trimethylol methane buffered saline ¾¾¾ of _9-14, ¾ water, sodium carbonate solution, or a solution of sodium hydrogen ¾.

The removal method according to claim 1, wherein the cation exchange material is a silica gel or a polymer material containing a sulfonic acid 3⁄4 a and/or a phosphoric acid group (poly-ene enoic acid) , polystyrene type 3⁄4, poly-ene amide type 3⁄4 quality; material" - is a granular material or a monolithic material.

7. The removal method according to claim 1, wherein: the contact and separation of the adsorbent and the sample solution are as follows:

Is the adsorbent immobilized on a solid support or filled in a hollow? In the device, will the sample solution containing the liquid pass through the ^ phase carrier or the towel empty? ,, and contact with the adsorbent Λ , direct collection;

8. The method of removing according to claim 6, wherein:

The phase carrier is: a surface area of 1 cm ² -K) m ² , and the shape is one or more of a K square, a cube, a cone or a cylinder, and the material is a polymer material, specifically a silica gel, a fiber* film, Polyether W film, resin, dextran gel, agarose gel and magnetic composite.

Empty? ? The device is: internal cavity diameter ^ small rust steel '笞' with a cross-section diameter of 50 μηι-5 cm, 20-1000 μΐ pipette tip, 1-200 ml | ≤ | phase extraction (SPE) '笞' , 1-200 ml syringe needle '笞', 50-500 μηι inner diameter capillary '笞' or syringe filter chamber.

9. The method according to claim 1, wherein: the sample solution is one of or a mixture of an egg phage sample, a liposome sample, and a sputum sample, and the method is used for egg n Analysis of qualitative samples, liposome samples, and sputum f samples.