WO2014198113A1 - 溶液样品中含长烷基链的离子液体的去除方法 - Google Patents

溶液样品中含长烷基链的离子液体的去除方法 Download PDF

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WO2014198113A1
WO2014198113A1 PCT/CN2013/090129 CN2013090129W WO2014198113A1 WO 2014198113 A1 WO2014198113 A1 WO 2014198113A1 CN 2013090129 W CN2013090129 W CN 2013090129W WO 2014198113 A1 WO2014198113 A1 WO 2014198113A1
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solution
adsorbent
liquid
sample
chain
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French (fr)
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张丽华
赵群
杨开广
方菲
张玉奎
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Dalian Institute of Chemical Physics of CAS
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F6/00—Post-polymerisation treatments
    • C08F6/06—Treatment of polymer solutions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N2001/4038—Concentrating samples electric methods, e.g. electromigration, electrophoresis, ionisation

Definitions

  • Solution sample 1 1 'method of removing ionic liquid containing long alkyl chain
  • 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 ⁇ 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.
  • 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 ⁇ is integrated on the membrane.
  • 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.
  • 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.
  • 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 ⁇ 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. .
  • the added solubilizer such as the f liquid will affect the mass spectrometry signal of the egg ⁇ mass analysis. Therefore, before mass spectrometry, it is necessary to remove the solubilizing agent from the Sin sample to obtain a mass spectrometric signal.
  • 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.
  • 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 ⁇ .
  • ⁇ the environment of pH ⁇ 8; then ⁇ 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 ⁇ ⁇ , N0 3 ⁇ C10 4 ⁇ A1C1 BF 4 ⁇ PF 4 ⁇ CF 3 CO(J, CF 3 SO" (CF 3 S0 2 ) 2 N- or SbF "
  • 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.
  • 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:
  • the adsorbent and the sample solution are mixedly contacted, and the adsorbent is separated from the solution containing the ex vivo by centrifugal precipitation;
  • the adsorbent is wrapped on the magnetic particles, and the adsorbent and the sample solution are mixed and contacted with ⁇ , 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.
  • the device is: internal cavity diameter ⁇ small rust steel ' ⁇ ' with a cross-section diameter of 50 ⁇ -5 cm, 20-1000 ⁇ pipette tip, 1-200 ml
  • SPE phase extraction
  • the sample solution is one of an egg ⁇ 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 .
  • 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?
  • 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 ⁇ L, 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
  • the adsorbent is loaded on the ⁇ phase carrier or filled in the towel?
  • the effluent is collected directly, that is, the solution of the effluent is removed; if the sorbent is not granulated by
  • 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.
  • *1 1 is the MALDI-TOF mass spectrometer of BSA hydrolysate.
  • Winter I 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.
  • Example 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 ⁇ ⁇ ⁇ : : : : : SUTi, pass Into a 30 mM bowl of acetamide (IAA) solution, room '; under the light-proof reaction for 30 min /Tiiit, the egg ⁇ alkane 3 ⁇ 4 treatment.
  • IAA acetamide
  • ⁇ according to an / pancreatic an enzyme: 25, add 4 ⁇ ⁇ Trypticase ⁇ enzyme (dissolved in 50 mM hydrogencarbonate hinge solution, pHS.O), digested for 12 hours at 37 ° C. Most; ⁇ , acidified with 10 ⁇ of formic acid, and finally hydrolyzed.
  • 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.
  • MALDI-TOF analysis the collected removal of the liquid from the f; ⁇ 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.
  • Nitrogen cleansing nSiri (BSA) sample pretreatment Dissolve the molar concentration of imgBSA in 1 ⁇ ⁇ 4 % ( 4 ⁇ ⁇ ) 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 ⁇ line ⁇ ⁇ Also 1 33 ⁇ 4 treatment; ⁇ , access to 30 mM bowl of acetamide (IAA) solution, chamber ';
  • MALDI-TOF analysis the collected removal of the f liquid; ⁇ BSA enzymatic solution solution frozen; Ti, fi dissolved in 500 ⁇ L 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; ⁇ , 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 / ⁇ at 90 ° C. Add 10 mM - sulfur « sugar alcohol (DTT) solution, 5 (60 ° TC reaction 60; 1!
  • 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 ⁇ ⁇ ⁇ 1 ⁇ 3 ⁇ 4 place: SUTi, pass 30 mM bowl of acetamide (IAA) solution, room ';

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Polymers & Plastics (AREA)
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  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
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Abstract

一种含长垸基链的离子液体的去除方法，包括以下步骤：a）将含有离子液体的溶液调至碱性（pH≥8）；b）采用阳离子交换材料作为吸附剂，去除含长垸基链的离子液体；c）收集去除离子液体后的溶液。该方法能很好地去除溶液中的含长垸基链的离子液体，具有简单、高效、快速的优点，并在蛋白质组学、高分子化学及脂质体代谢组学方面有着广阔的应用前景。

Description

溶液样品 11 '含长烷基链的离子液体的去除方法
技术领域
木发明涉及一种含 烷¾链的离 f液体的去除方法及其应用， "」-¾现溶液巾含 κ烷 ¾链的离 f液体的有效去除， 具有简^、 髙效、 快速的优点， 并 11该方法 以广泛应用 于蛋 n质组学、 髙分 f化学及脂质体代谢组学的研究巾。
背景技术
膜蛋 n质对执行细胞 A外物质交换、 细胞识别与免疫应答、 信号传导和调控以及能 M传递等功能起着 s要作用。 真核牛物巾 1/3的蛋 η均整合在膜上。 此外， 膜蛋 η质在 药物研究巾也起着相当 fi要的作用，在 知的和正在研究的药物靶标巾大约有 70%为膜 sin质。 然而， 山于膜蛋 质疏水性强， 导致其溶解性和酶解效率较差， 分析闲难。 因 此， 选择一种对膜 sin质具有髙溶解能力的溶剂是膜 sin质研究的¾决条件。
近年 *， 离 f液体作为一种极具应用前景的绿色溶剂受到越 *越多的关注。 离 体是完个山阴阳离 f组成， 通常在室温下表现为液体或是熔点小于 100 °c的盐类。 离 液体具有良好的溶剂性、 小挥发、 热稳定、 液程宽、 阴阳离 设计性等优点， 经 被广泛应用在化学合成、 电化学、 萃取分离、 材料制备等诸多领域。 山于其良好的溶解 能力， 近来年离 f-液体成为膜 ίίίΠ质溶解的新型溶剂 （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. 599, 3387-3397. ) 。
"Bottom-up"技术是 B前蛋 ri质组学分析鉴定巾最常用的技术， 该技术是¾将蛋 Π 质酶解成肽段， 然 通过一维或者多维分离技术 ϋΜϊ分离及在线质谱鉴定。 在膜蛋 Π质 分析巾， 为丫膜 sin质的增溶， 添加的离 f液体等增溶剂会严 s影响蛋 η质分析的质谱 信号。 因此质谱分析前， 需要对 Sin质样品 ϋΜϊ离 液体等增溶剂的去除， 以获得理 .《 的质谱信号。
发明内容
为丫解决上述 i。j题， 木发明的 B的在于发展一种含 烷¾链的离 液体的去除方 法， 通过该方法能髙效、 快速、 简便地去除溶液中的离 液体， 从而与 续的分析过程 相兼？？， 小对 续的分析造成小利的影响。
为¾现上述 B的， 木发明 ¾用的技术方案为：
溶液样品中含 烷¾链的离 f液体的去除方法， 将含有 烷¾链的离 f液体的样品 溶液用诚性溶液调. Φ: pH≥8的诚性环境； 然 Λ ¾用阳离 f¾换材料作为吸附剂， 去除含 烷¾链的离 f液体， 最 将吸附剂和溶液分离， 收集溶液。
含 烷¾链的离 液体 为： 阳离 部分为烷¾链部分， 为含 6个碳或 7个以上碳 的咪唑类、 吡啶类、 .手铰类、 或.手鳞类阳离 Λ 阴离 部分为 Cr、 Br\ Γ、 N03\ C104\ A1C1 BF4\ PF4\ CF3 CO(J、 CF3 SO" (CF3 S02) 2N-或 SbF "
将含有 K烷 ¾链的离 f液体的样品溶液用诚性溶液调. Φ: ρΗ为 8-14的诚性环境。 含有 烷¾链的离 液体的样品溶液 为：细胞提取液、胞浆提取液、血浆提取液、 蛋 质溶液、 多肽溶液、 脂质物溶液或 .电负性的聚合物溶液。
调节 ρΗ 诚性环境所 ¾用的诚性溶液为 ρΗ为 9-Μ的碳酸氢铰缓冲盐溶液、 ρΗ为 9-14的磷酸缓冲盐溶液、 ρΗ为 9-14的三羟甲 ¾¾¾甲烷缓冲盐溶液、 ¾水、 碳酸钠溶 液、 或氢¾化钠溶液。
阳离 J"交换材料为含有磺酸¾团和 /或磷酸¾团的硅胶或聚合物¾质材料（如：聚内— 烯酸《类¾质、 聚苯乙烯类 ¾质、 聚内-烯酰胺类 ¾质）； 材料 以是颗粒材料或者整体 材料。 将吸附剂和样品溶液接触和分离的方式 以为：
将吸附剂固载于固相载体上或填充于中空？?器中，将含有离 液体的样品溶液通过
^相载体或巾空？?器， 并与吸附剂接触 Λ ， 直接收集；
或将吸附剂和样品溶液混合接触 ， 将吸附剂通过离心沉淀的方式， 与含有离 体的溶液分离；
或将吸附剂包裹于磁性颗粒上， 将吸附剂和样品溶液混合接触 Λ ， 通过磁力作用， 与含有离 f液体的样品溶液分离。
^相载体为： 表面积为 lcm2-K)m2, 形状为 方体、 正方体、 圆锥体或圆柱体中一 种或 ：种以上， 材质为聚合物材料， 具体为硅胶、 纤维 *膜、 聚醚 膜、 树脂、 葡聚糖 凝胶、 琼脂糖凝胶和磁性复合材料。
巾空？？器为： 内部空腔径 ^横截面直径为 50 μηι-5 cm的小锈钢'笞'、 20-1000 μΐ移液 器枪头、 1-200 ml |≤|相萃取 (SPE)
Figure imgf000003_0001
1-200 ml注射器针'笞'、 50-500 μηι内径毛细'笞' 或注射器滤膜腔体。
所述样品溶液为蛋 η质样品、 脂质体样品及髙分 样品中一种或 -.种以上混合， ¾ 方法 」-用于蛋 n质样品、 脂质体样品及髙分 样品的分析中。
具体：
1、 将含有 fc«¾¾链的离 f液体的样品溶液， 用 pH为 9-M碳酸氢铰缓冲盐溶液、 磷酸缓冲盐溶液、 三羟甲 ¾¾¾甲烷缓冲盐溶液， ¾水、 碳酸钠溶液、 或氢¾化钠溶液 诚性溶液调. Φ: pH>8的诚性环境；
2、¾用含有磺酸¾团和 /或磷酸¾团的硅胶或聚合物¾质的阳离 交换材料作为吸 附剂， 吸附溶液中含 烷¾链的离 液体。 若吸附剂^载于^相载体上或填充于中空？? 器巾， 则将含 烷 ¾链离 f-液体的样品溶液以 1( 000 μL的流速直接通过吸附剂即 若吸附剂为未被^定化的颗粒， 则直接将吸附剂加入含 烷¾链离 f液体的溶液巾， 振 荡 3-10 min
3、 若吸附剂^载于^相载体上或填充于巾空？?器巾， 则直接收集流出液， 即为去 除离 f液体; ΓΪ的溶液；若吸附剂为未被 |≤|定化的颗粒， 则通过离心的方法（100-30 000g) 分离吸附剂和溶液. 收集溶液部分. 即为去除离 f液体 的溶液。
木发明具有如下优点：
1. 去除效率高。 pH>8的诚性范围 (A」， 绝大部分蛋 Π质或肽段成电负性， 山于所带 负电荷和离 f液体中含有 烷¾链的阳离 f的正电荷相反， ¾用强阳离 f¾换材料， 含 有 κ烷 ¾链的离 f液体被保留，而蛋 η质或肽段小保留， 从而将离 f液体和 si n质 /肽段 分开。
2. 操作方便、 快拢。 强阳离 交换材料只需和含 烷¾链的离 液体充分接触， 即 」-将 w者分离。随;^ ¾用离心或磁性分离等手段， 即 收去除离 液体 的溶液。 若强阳离 -交换材料被填装于柱钤 A或被^定化， 将含离 f液体的溶液通过阳离 -交换 材料， 收集流出液即为去除离 f液体 的溶液。
附图说明
*1 1为 BSA酶解产物的 MALDI-TOF质谱阁。 冬 I 1. 用强阳离 f交换材料为吸附剂（A) 将其填充于捕集柱 A (B)直接放入溶液巾，去除离 f液体 的 BSA酶解产物的质谱冬 I； (C) 小含离 f液体的 BSA酶解产物的质谱 ； (D) 含离 f液体的 BSA酶解产物的质 谱 。
具体实施方式
实施例 1 1. 牛血洁 Πίίίη (BSA) 样品预处理： 将 hngBSA溶解于 lmL4% (4g/100 mL) 的氯化 1-十 --烷 ¾-3-甲¾咪唑 （C12Im-Cl) 的 50 mM碳酸氢铰溶液 （ABC) 巾， 9(TC 下热变性 10min/Ti， 加入 10mM ：硫 «糖醇 （DTT) 溶液， 5(TC下反应 60 min ϋΜϊίίί Π质的还 1ί¾处: SUTi， 通入 30 mM碗乙酰胺 （IAA) 溶液， 室'； 下避光反应 30 min /Tiiit 行蛋 η质的烷¾化处理。 最; ΓΪ， 按 an质 /胰 an酶： 25 , 加入 4ομ§的胰蛋 η酶 （溶 于 50 mM碳酸氢铰溶液， pHS.O), 37°C酶解 12小时。 最; Γί， 加入 10 μΐ甲酸酸化， 终 ih酶解。
2. 含 烷¾链的离 液体的去除： 将强阳离 交换材料 （H木东曹达， TSK-GEL SP-5PW, 10 μηι, 1000 Α)填装于抛光小锈钢捕集柱柱 '笞'内 （内径 46 cm, K 1cm). ^摩尔浓度为 imgmL BSA的酶解产物 （ iOO μL, 溶于 4% C12Im-Cl的 50 mM ABC) 加入 400 μ1^ηιΜ三羟甲 ¾¾¾甲烷-盐酸缓冲盐溶液 （Tris-HCl, pH9.5)。 随; Γί， 将上 述 0.2 mg/mL BSA的酶解产物用 imM Tris-HCl, pH 9.5作为载流液， 以 im!Vmin的流 速， 用泵推动， 通过强阳离 换捕集柱， 收集流出液， 即为去除离 f液体 的溶液。
3. MALDI-TOF分析： 将收集的去除离 f液体； Γί的 BSA酶解产物溶液冻十; Ti, fi溶 于 SOO LO.P/o (体积分数）三¾乙酸水溶液， 即 0.2mgmLBSA酶解产物。 以
-4-羟¾肉桂酸 （7mgmL, 溶于含（U% (体积分数） 三¾乙酸的 60% 乙腊水溶液） 为 ¾质， 按体积比 1:1混合， 点样 2 L, Π然风十; Γί， MALDI-TOF检测。 质谱 1冬 I见 1冬 I 1 (A), 鉴定结果见表 i。
¾施例 2
1.牛血洁 nSiri(BSA)样品预处理：将摩尔浓度为 imgBSA溶解于 1ηΛ4%(4§ Κ)0 mL) 的氯化 1-十 ：烷 ¾-3-甲¾咪唑 （C12Im-Cl) 的 50 mM碳酸氢铰溶液 （ABC) 巾， 90°C下热变性 iOmin/Ti, 加入 10mM ：硫 «糖醇 （DTT) 溶液， 56°C下反应 60 min ϋί 行 ίίίΠ质的还 1ί¾处理； Γί， 通入 30 mM碗乙酰胺 （IAA) 溶液， 室'； 下避光反应 30 min
/ΓίϋΜϊίΐίΓΐ质的烷¾化处理。 最; Γί, 按 an质 /胰 an酶： 25A加入， 4ομ§的胰蛋 η酶 (溶于 50 mM碳酸氢铰溶液， pH 8.0), 37°C酶解 12小时。 最 fi, 加入 Κ) μΐ甲酸酸化， 终 ih酶解。
2. 含 K烷 ¾链的离 f-液体的去除： 14 lmgmLBSA的酶解产物 （K)0 L, 溶于 4%
C12Im-Cl 的 50 mM ABC) 加入 400 μL ImM 三羟甲 ¾¾¾甲烷 -盐酸缓冲盐溶液 (Tris-HCl, pH9.5)。 随; Γί， ^上述溶液中加入 2 mg强阳离 f-交换材料 (H木东曹达, TSK-GELSP-5PW, ΙΟμηι, 1000 Α),震荡 3min _/Γί,14000g离心 5min。 取上洁， 即为去 除离 f液体 的溶液。
3. MALDI-TOF分析： 将收集的去除离 f液体； Γί的 BSA酶解产物溶液冻十; Ti， fi溶 于 500 μL0.1% (体积分数）三¾乙酸水溶液， 即 0.2 mgmL BSA酶解产物。 以 -4-羟¾肉桂酸 （7mgmL, 溶于含（U% (体积分数） 三¾乙酸的 60% 乙腊水溶液） 为 ¾质， 按体积比 1:1混合， 点样 2 L, Π然风十; Γί， MALDI-TOF检测。 质谱 1冬 I见 1冬 I 1 (B), 鉴定结果见表 i。
对照组 1 用小含 K烷 ¾链的离 f-液体的溶剂溶解 BSA, ϋί行样品预处理： 将 img BSA溶解 于 imL 50 mM碳酸氢铰溶液 （ABC) 巾， 90°C下热变性 min /Γί， 加入 10 mM --硫 «糖醇 （DTT) 溶液， 5(TC下反应 60 ；1!1；行蛋|"1质的还原处理；：；， 通入 30 mM碗乙 酰胺 （iAA) 溶液， 室'； 下避光反应 ； ；1!1；行蛋1^质的烷¾化处理。 i, 按 an 质 /胰蛋 Π酶： 25A, 加入 40μ§的胰蛋 Π酶（溶于 50 mM碳酸氢铰溶液， pHS.O), 3TC 酶解 12小时。最; Γί,加入 10 μΐ甲酸酸化，终 lh酶解。将 BSA酶解产物稀释 0.2 mg/mL, 以 2-«¾-4-羟¾肉桂酸 （7mgmL, 溶于含 O.P/。 （体积分数） 三¾乙酸的 60% 乙腊水 溶液） 为¾质， 按体积比 1:1混合， 点样 2 L, Π然风十； Γί， MALDI-TOF检测。 质谱 1冬 I见 1 1 (C), 鉴定结果见表 1。
对照组 2
用含 烷¾链离 f液体的 BSA酶解产物溶液为对照组：将 lmg BSA溶解于 imL 4% (4g100 mL)的氯化 i -十 ：烷 ¾-3-甲¾咪唑（C12Im-Cl)的 50 mM碳酸氢铰溶液（ABC) 巾， 90°C下热变性 iOmin/Ti, 加入 10 mM -.硫 «糖醇（DTT)溶液， 56°(：下反应601 ϋΜϊίίίΓΐ质的还 1ί¾处: SUTi， 通入 30mM碗乙酰胺（IAA)溶液， 室'； 下避光反应 30min
/ΓίϋΜϊίΐίΓΐ质的烷¾化处理。 最; Γί, 按 an质 /胰 an酶： 25A, 加入 4ομ§的胰蛋 η酶
(溶于 50 mM碳酸氢铰溶液， pH8.0), 37°C酶解 12小时。 最 fi, 加入 10 μΐ甲酸酸化， 终 lh酶解。 将 BSA酶解产物稀释 0.2 mg/mL, 以 2-¾¾-4-羟¾肉桂酸 （7mgmL, 溶 于含 (U% (体积分数）三¾乙酸的 60% 乙腊水溶液） 为¾质， 按体积比 1:1混合， 点 样 2 L, Π然风十； Γί， MALDI-TOF检测。 质谱 1冬 I见 1冬 I 1 (D), 鉴定结果见表 L
表 L BSA酶解产物的鉴定结果
Figure imgf000005_0001
通过表 1和 1 1 以 出， 通过该发明的方法， 含有 烷¾链的离 液体 以从溶 液中髙效去除， 从而小影响质谱的检测。

Claims

权 利 要 求 书
1. 溶液样品中含 烷¾链的离 f液体的去除方法， 其特征在于： 将含有 烷¾链 的离 f液体的样品溶液用诚性溶液调. Φ: pH>8的诚性环境； 然 Λ ¾用阳离 f交换材料作 为吸附剂， 去除含 烷¾链的离 液体， 最 将吸附剂和溶液分离， 收集溶液。
2. 根据权利要求 1所述的去除方法， 其特征在于： 含 K烷 ¾链的离 f液体 为： 阳离 部分为烷¾链部分， 为含 6个碳或 7个以上碳的咪唑类、 吡 类、 .手铰类、 或.手 it类阳离 Λ 阴离 f部分为 C 、 Br\ Γ、 Ν(¾·、 C104\ A1C14\ BF4\ PF4\ CF3 COO"、 CF3 S03\ (CF3 S02)2N-或 SbF 6-。
3. 根据权利要求 1所述的去除方法， 其特征在于： 将含有 K烷 ¾链的离 f液体的 样品溶液用诚性溶液调. Φ: pH为 8-14的诚性环境。
4. 根据权利要求 1所述的去除方法， 其特征在于： 含有 烷¾链的离 f液体的样 品溶液 为： 细胞提取液、 胞浆提取液、 血浆提取液、 sin质溶液、 多肽溶液、 脂质物 溶液或 .电负性的聚合物溶液。
5. 根据权利要求 1所述的去除方法， 其特征在于： 调节 ρΗ .Φ:诚性环境所 ¾用的诚 性溶液为 pH为 9-14的碳酸氢铰缓冲盐溶液、 pH为 9-14的磷酸缓冲盐溶液、 pH为 9-14 的三羟甲 ¾¾¾甲烷缓冲盐溶液、 ¾水、 碳酸钠溶液、 或氢¾化钠溶液。
6. 根据权利要求 1所述的去除方法， 其特征在于： 阳离 换材料为含有磺酸 ¾ a和 /或磷酸¾团的硅胶或聚合物¾质材料（聚内―烯酸《类¾质、聚苯乙烯类 ¾质、聚内— 烯酰胺类¾质）； 材料 」-以是颗粒材料或者整体材料。
7. 根据权利要求 1所述的去除方法， 其特征在于： 将吸附剂和样品溶液接触和分 离的方 OJ以为：
将吸附剂固载于固相载体上或填充于中空？?器中，将含有离 液体的样品溶液通过 ^相载体或巾空？?器， 并与吸附剂接触 Λ ， 直接收集；
或将吸附剂和样品溶液混合接触 ， 将吸附剂通过离心沉淀的方式， 与含有离 体的溶液分离；
或将吸附剂包裹于磁性颗粒上， 将吸附剂和样品溶液混合接触 Λ ， 通过磁力作用， 与含有离 f液体的样品溶液分离。
8. 根据权利要求 6所述的去除方法， 其特征在于：
^相载体为： 表面积为 lcm2-K)m2, 形状为 K方体、 正方体、 圆锥体或圆柱体中一 种或 ：种以上， 材质为聚合物材料， 具体为硅胶、 纤维 *膜、 聚醚 W膜、 树脂、 葡聚糖 凝胶、 琼脂糖凝胶和磁性复合材料。
巾空？？器为： 内部空腔径 ^横截面直径为 50 μηι-5 cm的小锈钢'笞'、 20-1000 μΐ移液 器枪头、 1-200 ml |≤|相萃取 (SPE) '笞'、 1-200 ml注射器针'笞'、 50-500 μηι内径毛细'笞' 或注射器滤膜腔体。
9、 根据权利要求 1所述的去除方法， 其特征在于： 所述样品溶液为蛋 η质样品、 脂质体样品及髙分 样品中一种或 -.种以上混合， 该方法 用于蛋 n质样品、 脂质体样 品及髙分 f样品的分析中。
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