WO2009044903A1 - Method of preparing protein sample - Google Patents

Method of preparing protein sample Download PDF

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Publication number
WO2009044903A1
WO2009044903A1 PCT/JP2008/068124 JP2008068124W WO2009044903A1 WO 2009044903 A1 WO2009044903 A1 WO 2009044903A1 JP 2008068124 W JP2008068124 W JP 2008068124W WO 2009044903 A1 WO2009044903 A1 WO 2009044903A1
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Prior art keywords
surfactant
protein
sodium
acid
organic solvent
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PCT/JP2008/068124
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French (fr)
Japanese (ja)
Inventor
Yasushi Ishihama
Takeshi Masuda
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Keio University
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Priority to JP2009536129A priority Critical patent/JP4831708B2/en
Publication of WO2009044903A1 publication Critical patent/WO2009044903A1/en

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    • 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
    • G01N1/4055—Concentrating samples by solubility techniques
    • 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
    • G01N1/4044—Concentrating samples by chemical techniques; Digestion; Chemical decomposition

Definitions

  • the present invention relates to a method for preparing a protein sample, for example, an insoluble protein sample for use in mass spectrometry.
  • Non-Patent Document 1 discloses a technique using sodium deoxycholate (hereinafter also referred to as “SDC”) as a technique for removing a surfactant after digestion of a membrane protein.
  • SDC sodium deoxycholate
  • This document states that SDC at a maximum concentration of 20% does not significantly affect trypsin activity, and that SDC is precipitated by adding trifluoroacetic acid to a protein digestion reaction solution containing SDC. It describes that SDC can be removed by centrifugation prior to analysis.
  • Non-Patent Document 2 shows that the use of an acid-labile surfactant in a trypsin digestion buffer increases protein solubilization and degradation, and trypsin digestion. It describes that acid labile surfactant can be removed by centrifugation after acidifying the buffer.
  • Non-Patent Literature 1 Zhou et al., Journal of Proteome Research 2006, 5,
  • Non-Patent Document 2 Chen et al., Journal of Proteome Research 2007 Jul; 6 (7): 2529-38 “0ptimization of Mass Spectrometry- Compatible Surfactants for Shotgun Proteomics
  • the method of precipitating and removing the surfactant after digestion of the protein sample as described above can avoid the problem of contamination of the analyzer by the surfactant, but the recovery efficiency of the protein digestion fraction is Not enough. This is thought to be due to the loss of some protein fractions during solid-liquid separation of the surfactant precipitate that occurs in the digestion reaction solution.
  • the present invention can sufficiently digest the protein to be analyzed while avoiding the problem of contamination of the analyzer due to the surfactant, and can sufficiently recover the digested protein or peptide fragment.
  • An object of the present invention is to provide a method for preparing a protein sample.
  • the surfactant is fat-solubilized and separated into an organic solvent.
  • the present invention includes the following features.
  • a method for preparing a protein sample comprising separating the fat-solubilized surfactant in an organic solvent.
  • Carboxylic acid type surfactants are sodium cholate, glycocholic acid, sodium lauryl sarcosinate, sodium deoxycholate, sodium glycochenodeoxycholate, sodium kenodeoxycholate or sodium ursodeoxycolate. (5) The method described in (5) above.
  • Acid labile surfactants include 3-[(2-methyl-2-undecyl-1,3-dioxol-4-yl) methoxyl] -1-propanesulfonic acid sodium or 3- [3- It is characterized by being (1, 1-alkyloxychetyl) pyridine-1-yl] propane-1-sulfonic acid
  • reaction solution is in the form of microemulsion.
  • step (b) The method according to (1), wherein after step (b), the organic solvent is added to the reaction solution, and the fat-solubilized surfactant is separated into the organic solvent.
  • a protein analysis method comprising preparing a protein sample by the method according to any one of (1) to Q7) and subjecting the sample to analysis.
  • Figure 1 shows a comparison of the protein identification results of the E. coli membrane fraction in the phase transfer method, acid precipitation method, and urea method using SDC.
  • Fig. 2 shows that when 10% sodium dodecyl sulfate (SDS) is used as a surfactant and double dose of octanol (OctOH) is used as an organic solvent in the present invention, the fat-solubilized SDS is used. It shows that liquid-liquid separation was complete.
  • SDS sodium dodecyl sulfate
  • OctOH octanol
  • Figure 3 shows the complete fat-solubilized sodium cholate in the present invention when 10% sodium cholate was used as the surfactant and doubled octanol (OctOH) was used as the organic solvent for the digestion reaction solution. Shows liquid-liquid separation.
  • FIG. 4 shows that when 10% glycocholic acid is used as a surfactant and octanol (OctOH) is used as an organic solvent in an amount equal to that of a digestion reaction solution in the present invention. It shows that lycocholic acid was completely liquid-liquid separated.
  • OctOH octanol
  • FIG. 5 shows that when 1% sodium lauryl sarcosinate is used as a surfactant and 0.5 times the amount of octanol is used as an organic solvent in the present invention, the fat-solubilized lauryl sarcosine acid is used. This shows that sodium was completely liquid-liquid separated.
  • FIG. 6 shows that in the present invention, when 10% sodium deoxycholate is used as a surfactant and 0.5 times the amount of octanol is used as an organic solvent with respect to the digestion reaction solution, the fat-solubilized sodium deoxycholate is completely liquid. Indicates that the liquid could be separated.
  • FIG. 7 shows that in the present invention, when 10% sodium deoxycholate is used as the surfactant and hexanol equivalent to the digestion reaction solution is used as the organic solvent, the fat-soluble sodium deoxycholate is completely liquid-liquid separated. Indicates that it was possible.
  • FIG. 8 shows that in the present invention, when 1% sodium deoxycholate is used as a surfactant and ethyl acetate equivalent to the digestion reaction solution is used as an organic solvent, the fat-solubilized sodium deoxycholate is completely liquid-liquid separated. It shows that it was possible.
  • FIG. 9 shows that in the present invention, 1% sodium deoxycholate was used as a surfactant, and 6 times the amount of jetyl ether was used as an organic solvent for the digestion reaction solution. Indicates that liquid-liquid separation was possible.
  • Fig. 1 ⁇ shows that in the present invention, 1% sodium deoxycholate was used as the surfactant, and 8 times the dose of black mouth form with respect to the digestion reaction solution was used as the organic solvent. Shows that liquid-liquid separation was possible.
  • Figure 1 in the present invention, the l% RapiG eS t TM SF as a surfactant, when using Otatanoru of 0.5 times the dose to digestion reaction solution as the organic solvent, Abura ⁇ of RapiGest TM SF Indicates that liquid-liquid separation could be completed.
  • Fig. 12 shows the fat-solubilization in the present invention when 1% glycochemodeoxychol sodium is used as a surfactant and 0.5 times the amount of octanool as an organic solvent is used as a digestion reaction solution. Completely liquid sodium daricochenodeoxycholate This shows that liquid separation was possible.
  • Fig. 13 shows the fat solubilization in the present invention when 10% sodium kenodeoxycholate was used as the surfactant and 1.5 times the amount of octanol was used as the organic solvent for the digestion reaction solution. This shows that sodium kenodeoxycholate was completely liquid-liquid separated.
  • Fig. 14 shows fat-solubilized urso when 10% sodium ursodeoxycholate is used as a surfactant and 3.5 times the dose of octanol is used as an organic solvent in a digestion reaction solution. This shows that sodium deoxycholate was completely liquid-liquid separated.
  • Fig. 15 shows a comparison of protein identification results of E. coli membrane fractions by the phase transfer method using SDC, SLS and SDC-SLS mixed systems.
  • the numbers in the figure indicate the total number of proteins identified from the E. coli membrane fraction, and the numbers in Katsuko indicate the number of membrane proteins identified.
  • Figure 16 shows a comparison of the number of transmembrane domains in the identified membrane proteins of the E. coli membrane fraction by phase transfer using SDC, SLS and SDC-SLS mixed systems.
  • the surfactant used for solubilizing the protein is modified to be fat-soluble, thereby separating the surfactant into an organic solvent (hereinafter,...
  • phase transfer method it is possible to remove the surfactant, which is likely to affect the analyzer, from the sample containing the protein fraction after the protease treatment.
  • the problem of partial deletion of protein fractions due to the formation of surfactant precipitates can be avoided, and this has the advantage of improving the protein recovery efficiency.
  • the method of the present invention comprises: (a) (i) a protein to be analyzed; (ii) a surfactant that can be modified to be lipophilic; and (iii) a protein to be analyzed in a reaction solution containing a protease. And (b) adding a reagent for fat-solubilizing the surfactant to the reaction solution.
  • step (a) the protein to be analyzed is added to a solution containing a surfactant that can be modified to be lipophilic. It is a step of dissolving the quality and digesting the protease.
  • the protein to be analyzed is not particularly limited, and for example, tissues, biological fluids, cells, pulverized organs, protein extracts and the like can be prepared and used according to conventional methods.
  • the protein to be analyzed includes a membrane protein, a protein having undergone post-translational modification such as GPI anchor (daricosylphosphatidylinositol anchor), or an insoluble or hardly soluble protein such as a membrane-attached protein. It is particularly effective for
  • the “surfactant that can be modified to be lipophilic” refers to a surfactant that increases in whole or part of its lipophilicity due to, for example, a change in PH caused by the addition of a specific reagent.
  • examples of the surfactant that can be modified to be lipophilic include an ionic surfactant and an acid labile surfactant.
  • the cationic surfactants that can be used in the present invention include both cationic surfactants and anionic surfactants.
  • a carboxylic acid type surfactant that is modified to be lipophilic under acidic conditions as the ionic surfactant.
  • carboxylic acid type surfactants include, but are not limited to, sodium cholate, sodium deoxycholate, glycocholic acid, sodium glycochenodeoxycholate, chenodeoxycol.
  • carboxylic acid type surfactants include, but are not limited to, sodium cholate, sodium deoxycholate, glycocholic acid, sodium glycochenodeoxycholate, chenodeoxycol.
  • cholic acids such as sodium acid sodium and sodium sodium sodium sodium hydroxysuccinate
  • sarcosines such as sodium lauryl sarcosinate
  • amino acid derivatives obtained by alkylating amino groups include sodium lauryl sarcosinate.
  • a sulfuric acid type surfactant as the ionic surfactant.
  • a sulfuric acid type surfactant include sodium dodecyl sulfate (SDS) which can be modified to be lipophilic by adding force lime.
  • an acid labile surfactant refers to a surfactant that acquires lipophilicity by separation of the hydrophobic part in the intramolecular structure under acidic conditions.
  • the hydrophilic part and hydrophobicity under acidic conditions It may be ionic or nonionic as long as it can be cut into parts. In view of protein solubilization ability, it is preferable to use an ionic acid labile surfactant.
  • Such acid labile surfactants include 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-inole) methoxyl] -1-propanesulfone Sodium acetate, 3_ [3- (1,1-alkyloxetyl) pyridine-1-yl] propane-1-sulfonic acid, etc., and trade names Rapigest TM SF (Water Corporation, Milford, MA ), PPS SILENT TM (Protein Discovery, Inc., Knoxville, TN).
  • Rapigest TM SF and PPS SILENT TM are described below:
  • the above-mentioned commercially available acid labile surfactant can be used as the acid labile surfactant.
  • examples of the surfactant that can be used in step (a) include a zwitterionic surfactant.
  • a zwitterionic surfactant is a surfactant that, when dissolved in water, exhibits the properties of an anionic surfactant in the alkaline region and the properties of a cationic surfactant in the acidic region, including but not limited to CHAPS, Examples include 3-[(3-choramidopropyl) dimethylan-o] -2-hydroxypropanesulfonic acid (CHAPS0), alkylbetaines, and the like.
  • the concentration of the surfactant added to the protease digestion reaction solution depends on the field of use. Although it varies depending on the surfactant, it is not particularly limited as long as it does not inactivate the protease. Therefore, it has the advantage that it can be used at a higher concentration than the concentration of the surfactant that has been conventionally used. That is, the phase transfer method of the present invention does not cause the problem of partial deletion of the protein fraction caused by the precipitate formation of the surfactant recognized in the prior art. As a result, it is possible to solubilize highly hydrophobic proteins that could not be identified in the past. This makes it possible to digest even highly hydrophobic proteins with protease.
  • the concentration of the surfactant added is 0.01 to 20% (w / v), preferably 0.01 to 15% (w / v), more preferably 0, depending on the surfactant used. 01-10% (w / v), for example 1% (w / v).
  • a person skilled in the art can appropriately set the optimum surfactant concentration that does not inactivate the protease according to the type of surfactant used.
  • the concentration of the surfactant is 0.5 to 20 times the amount of the digestion reaction solution after fat solubilization, preferably 0.5 to 15 times the dose, more preferably 0.5 to 10 times the amount of the organic solvent.
  • the concentration is preferably dissolved.
  • the protease used in step (a) is not particularly limited, and those skilled in the art can appropriately select a protease according to the purpose of protein digestion.
  • trypsin is preferably used as a protease.
  • Step (b) is a step of solubilizing the surfactant used in step (a).
  • a reagent for fat-solubilizing the surfactant used in step (a) is added to the protease digestion reaction solution.
  • the reagent to be added depends on the surfactant used in step (a), but is typically an acid or potassium salt.
  • the form of the acid that can be used in step (b) is not particularly limited, and examples thereof include trifluoroacetic acid (TFA), hydrochloric acid, sulfuric acid, phosphoric acid, hexafluorobutyric acid, acetic acid, formic acid and the like. .
  • the amount added is the protease digestion reaction It is an amount sufficient to make the pH of the solution acidic, that is, pH 0-4, preferably pH 0-3. Depending on the amount of acid added, at least 50%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the surfactant in the protease digestion reaction solution. It is preferable to change to fat solubility.
  • the form of the potassium salt that can be used in step (b) is also not particularly limited.
  • any potassium salt form such as potassium chloride, potassium dihydrogen phosphate, potassium acetate, potassium formate can be used.
  • the amount of addition varies depending on the concentration of the surfactant (eg, SDS) contained in the protease digestion reaction solution, but is at least 50%, preferably less than the surfactant contained in the reaction solution. It is preferred to set the amount to 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95% modified to be fat-soluble.
  • the surfactant can be separated into the organic solvent by solubilizing the surfactant used in step (a).
  • the organic solvent may be added in advance to the protease digestion reaction solution, or may be added simultaneously with or after the addition of the reagent that solubilizes the surfactant.
  • the solution is preferably in the form of an emulsion.
  • Emulsion refers to a state where oil droplets are dispersed in water, and in this state, the surface area of the oil droplets in contact with water is increased. Therefore, it has the advantage of making the protein easier to dissolve.
  • An emulsion can be easily prepared by vigorously shaking a solution containing water and an organic solvent.
  • the protease digestion reaction solution takes the form of a microemulsion.
  • microemulsion refers to those in which the particle size of oil droplets is about 10 im, even among the emulsion openings. Microemulsions have the advantage that the surface area of the oil droplets in contact with water is larger than that of normal emulsion, and the solubility of the protein can be further increased. In addition, proteases are not affected when the digestion reaction solution is in the form of a microemulsion. It has been confirmed that it is excellent in maintaining protease activity. Furthermore, unlike ordinary emulsions, microemulsions have the advantage that they can remain in their form for several days even when they are stationary.
  • Microemulsions can be produced by mixing water, an organic solvent, a surfactant, and optionally an auxiliary surfactant in a predetermined range in a protease digestion reaction solution, and performing ultrasonic treatment or the like.
  • an organic solvent is added at the same time as the reagent that solubilizes the surfactant, or after the addition of the reagent, the organic solvent is added after the protease treatment.
  • the organic solvent that can be used in the method of the present invention is not particularly limited as long as it can be separated into water and two phases.
  • octanol ethyl acetate, jetyl etherol, chlorohonolem, hexanol, heptanol monore , Dichloromethane, dioxane, benzene, toluene, hexane, heptane, octane, pyridine and the like.
  • the amount of the organic solvent to be added can be appropriately determined by those skilled in the art depending on the type and concentration of the surfactant to be used and the type of the organic solvent to be used.
  • the organic solvent is in an amount that dissolves at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the fat-solubilized surfactant. It is preferable to add.
  • two or more kinds of surfactants that can be modified to be lipophilic can be used in combination. The use of two or more surfactants can further improve the recovery efficiency of protein digested fractions after protease treatment even at lower surfactant concentrations than using a single surfactant. There is an advantage that more proteins and membrane proteins can be identified.
  • the two or more surfactants may be selected from the same surfactant group or may be selected from different surfactant groups. Therefore, the two or more surfactants can be selected from the group consisting of ionic surfactants, acid labile surfactants and zwitterionic surfactants. Preferably, the two or more surfactants are selected from the same group of surfactants. As described above, since the reagent used for fat-solubilizing the surfactant differs depending on the nature of the surfactant used, two or more surfactants belonging to the same surfactant group are used. This is because the complexity of adding a plurality of reagents can be avoided.
  • two or more surfactants are two or more selected from carboxylic acid type surfactants or two or more selected from acid labile surfactants, these surfactants It is only necessary to add an acid in order to fat-solubilize the agent.
  • the two or more surfactants are a combination of a carboxylic acid type surfactant and an acid labile surfactant that are modified to be lipophilic under acidic conditions.
  • a particularly preferred surfactant combination is a combination of sodium deoxycholate and sodium lauryl sarcosinate.
  • the method of the present invention can be applied to the digestion of proteins in gels.
  • the method of the present invention can be used in the process of digesting and extracting a protein contained in a gel cut out after separation by electrophoresis.
  • a surfactant is allowed to coexist in the protease solution used for in-gel digestion, and the pH of the extracted solution is neutralized during the extraction process from the gel after completion of in-gel digestion.
  • the peptide can be recovered with high efficiency by adding an organic solvent to the solution containing the peptide and the surfactant after the alkaline treatment, and shifting the solution to the acidic side as in the case of solution digestion.
  • the electrophoresis used is not particularly limited as long as the protein can be separated by electrophoresis through a gel and the molecular weight is different due to the difference in isoelectric point.
  • Examples include SDS-PAGE, isoelectric focusing, and two-dimensional electrophoresis. be able to. As a result, the protein expression efficiency, which is relatively low in expression level and difficult to identify by conventional methods, can be improved.
  • a highly hydrophobic protein such as a membrane protein can be sufficiently solubilized and treated with protease, and the surfactant is separated. Even so, the protein fraction after digestion can be recovered with high efficiency.
  • an example it is possible to prepare a protein sample that can reliably analyze a protein to be analyzed while sufficiently removing a surfactant that contaminates various analyzers such as a mass spectrometer.
  • the protein sample prepared by the method of the present invention can be subjected to any analysis such as mass spectrometry, amino acid analysis, and immunoassay using an antibody.
  • mass spectrometry a protein fraction that could not be identified by a conventional method can be detected and identified. That is, according to the method of the present invention, when a protein fraction is identified by an analysis system such as mass spectrometry, the identification efficiency can be greatly improved as compared with the conventional method.
  • LC-MS liquid chromatography mass spectrometry
  • MALDI-MS matrix-assisted laser desorption / ionization mass spectrometry
  • Example 1 Comparison of protein identification results in the phase transfer method, acid precipitation method and solution digestion method using urea of the present invention
  • a protein membrane fraction was used as an insoluble protein in order to investigate the solubilization and degradation efficiency of the insoluble protein.
  • the membrane fraction was collected from E. coli K-12 strain BW25113.
  • BW25113 was cultured in 50 mL of LB medium (37 ° C, 8 hours) and collected by centrifugation for 10 minutes (5000 rpm, 4 ° C).
  • the collected BW25113 was suspended in 5 mL of 1M potassium chloride and 10 mM Tris solution (washing buffer), and washed by centrifugation (5000 rpm, 4 ° C) for 10 minutes. Washing was performed twice.
  • BW25113 suspended in 5 mL of washing buffer was sonicated with an ultrasonic crusher (T0MY,
  • membrane protein solubilizers 0.5% sodium deoxycholate (D0JIND0 Cat. No. D520: SDC) and 8M urea (Wako Pure Chemicals Cat. No. 217-00615) were used.
  • 500 // L solubilizer was added and suspended, and then heated at 95 ° C for 5 minutes.
  • the membrane protein suspension was treated with an ultrasonic generator (T0CH0, UC-1331N) for 10 minutes, and the insoluble matter was removed by centrifugation at 15,000 rpm for 10 minutes.
  • T0CH0, UC-1331N ultrasonic generator
  • the above protein sample solution solubilized with 0.5% SDC was digested by adding 0.5 mg / mL trypsin (Promega, Cat. No. V511C) and incubating at room temperature for 24 hours. SDC was removed by acid precipitation or phase transfer. In the acid precipitation method, TFA was added to the digested sample to a final concentration of 1%, and insoluble SDC was removed by centrifugation at 13,000 rpm for 2 minutes. In the phase transfer method, an equal amount of ethyl acetate (Wako Pure Chemicals Cat. No. 055-05991) was added to the sample, and TFA was added to a final concentration of 1%.
  • the sample solution prepared by the treatments 1) and 2) was subjected to LC-MS measurement as follows.
  • C18-StageTip (homemade, J. Rappsilber, Y. Ishihama, M. Mann, Anal Chem 75 (2003) 663) was prepared using a 200 L pipette tip and an Empore C18 disk. StageTip is loaded with 20 L of methanol, 20 ⁇ L of 80% acetonitrile, 0.1% TFA (hereinafter referred to as “solution”) and 5% acetonitrile, 0.1% TFA (hereinafter referred to as “solution A”). Activated. Each sample solution prepared by the above processes 1) to 2) was loaded onto StageTip. After washing with 20 ⁇ L of solution, the peptide was eluted from StageTip with 40 L of solution B. The eluted peptide solution was concentrated by centrifugation and then dissolved in 20 ⁇ L of solution to prepare a sample solution for LC-MS.
  • HPLC conditions include C18 Siri-Force Gel (ReproSil-Pur 120 C18-AQ,
  • the sample solution was injected 5 ⁇ m by CTC autosampler PAL, and the sample was once injected into the sampler loop of the injector and then fed into the analytical ram.
  • the measurement was performed in Information dependent acquisition mode, after a 1-second survey scan, followed by a maximum of 3 MSMS scans (0.6 seconds each). The switch from the MSMS mode power to the survey scan was 1 spectrum.
  • the method using SDC combined with the phase transfer method increases the number of identified membrane proteins by about 3 times (SDC: 14, urea: 4). Overall, the number of identifications increased 5.08 times (SDC: 108, urea: 37). When comparing the number of peptides that could be identified, SDC increased approximately 5.3 times compared to urea.
  • phase transfer method of the present invention is greatly improved in terms of the number of proteins to be identified and the number of peptides as compared to the protein sample prepared by using the conventional method.
  • Example 1 liquid-liquid separation when a combination of various surfactants and organic solvents was used in the method of the present invention was evaluated.
  • the protein membrane fraction prepared in Example 1 was used as an insoluble protein sample, and the same procedure as in Example 1 was performed except for the points described below.
  • Surfactant Organic solvent Combination 1 10% sodium dodecyl sulfate (SDS) Octanol (OctOH) Combination 2. 10 ⁇ /. Sodium cholate octanol combination 3. 10% glycocholic acid pentanol combination 4. 1% and 10% sodium lauryl sarcosinate octanol combination 5. 10% sodium deoxycholate octanol combination 6. 10% sodium deoxycholate hexanol combination 7.1 % And 10% Sodium Deoxycholate Ethyl Acetate (EtAc) Combination 8.1% and 10% Sodium Deoxycholate Jetinoreethenole Combination 9. 1% and 10% Sodium Deoxycholate Chlorophore / REM Combination 10.
  • the SDS salt was exchanged with 50 uL of 1M KC1, 15m Tris.
  • RapiGest TM SF was added to 0.5% TFA and incubated for 30 minutes at 37 ° C to degrade.
  • octanol as the organic solvent is equivalent to the digestion reaction solution except when 10% sodium lauryl sarcosinate or 10% sodium daricochenodoxycholate is used as a surfactant. It was possible to completely dissolve the fat-solubilized surfactant in 5 times the dose of octanol ( Figures 2-5, 13-14). These results indicate that the use of octanol as the organic solvent is relatively suitable for dissolving high concentrations of fat-solubilized surfactants.
  • Example 3 Comparison of protein identification results in solution digestion using sodium deoxycholate, sodium lauryl sarcosinate and mixtures thereof in the phase transfer method of the present invention
  • the membrane fraction was prepared from E. coli K-12 strain BW25113 in the same manner as in Example 1. 120 raM (equivalent to 5%) sodium deoxycholate for membrane protein solubilizer

Abstract

It is intended to provide a method of preparing a protein sample whereby a protein to be analyzed can be sufficiently digested enzymatically and the digested protein or peptide fragments can be sufficiently collected while avoiding the risk of the contamination of an analyzer with a surfactant. A method of preparing a protein sample characterized in that, in the technique of solubilizing a protein by using a surfactant and then digesting with a protease followed by the removal of the surfactant, the surfactant is made fat-soluble and separated into an organic solvent.

Description

タンパク質試料の調製方法 技術分野  Protein Sample Preparation Method Technical Field
本発明は、 タンパク質試料、 例えば、 質量分析に使用するための不溶性タンパ ク質試料の調製方法に関する。 明  The present invention relates to a method for preparing a protein sample, for example, an insoluble protein sample for use in mass spectrometry. Light
背景技術 Background art
田  Rice field
タンパク質を質量分析計で測定する場合、 感度などの問題からトリプシンなど の切断特異性の明らかな消化酵素で断片化した後に測定する。 その際、 溶液中で 消化する場合と、 ゲル電気泳動で分離後、 ゲル内で消化する場合があり、 それぞ れに一長一短があるが、 これらの方法によりプロテオームレベルでのタンパク質 の網羅的な解析が可能となっている。  When measuring a protein with a mass spectrometer, it is measured after fragmentation with a digestive enzyme with a clear cleavage specificity such as trypsin due to problems such as sensitivity. At that time, there are cases of digestion in solution and separation by gel electrophoresis, followed by digestion in the gel. Each has advantages and disadvantages, but these methods comprehensively analyze proteins at the proteome level. Is possible.
タンパク質を消化酵素で断片化する際には、 タンパク質を可溶化する必要があ る。 従来、 タンパク質、 特に不溶性タンパク質を可溶化するために、 界面活性剤 や有機溶媒などが使用されてきた。 しかし、 有機溶媒はタンパク質可溶化能に乏 しく、 高濃度で添加すると逆にタンパク質を不溶化したり、 消化酵素を変性させ てしまうという不都合を有する。 また界面活性剤も消化酵素の変性という問題を 有する上、 分析装置の汚染の問題を抱えている。  When a protein is fragmented with a digestive enzyme, it is necessary to solubilize the protein. Conventionally, surfactants and organic solvents have been used to solubilize proteins, particularly insoluble proteins. However, organic solvents have poor ability to solubilize proteins, and if added at a high concentration, they have the disadvantage that they insolubilize proteins and denature digestive enzymes. Surfactants also have the problem of digestive enzyme denaturation and the problem of contamination of the analyzer.
このような状況において、 近年、 タンパク質試料を可溶化するために界面活性 剤を用い、 タンパク質試料の消化後に界面活性剤を除去する技術が提案されてい る。  Under such circumstances, in recent years, a technique has been proposed in which a surfactant is used to solubilize a protein sample and the surfactant is removed after digestion of the protein sample.
例えば、 非特許文献 1は、 膜タンパク質の消化後に界面活性剤を除去する技術 として、 デォキシコール酸ナトリウム （以下、 「SDC」 とも称する） を用いる手法 を開示している。 この文献には、 最大 2, 0%濃度の SDCはトリプシンの活性に有 意な影響を与えないこと、 及び SDCを含むタンパク質消化反応溶液にトリフルォ 口酢酸を添加することで SDCを沈殿させ、 質量分析前に SDCを遠心分離除去でき ることが記載されている。 P T/JP2008/068124 また非特許文献 2は、 トリプシン消化バッファ一中での酸不安定界面活性剤 (acid-labile surfactant) の使用が、 タンパク質の可溶化及び分解を増加させ ること、 及びトリプシン消化バッファーを酸性化した後に酸不安定界面活性剤を 遠心分離除去できることを記載している。 For example, Non-Patent Document 1 discloses a technique using sodium deoxycholate (hereinafter also referred to as “SDC”) as a technique for removing a surfactant after digestion of a membrane protein. This document states that SDC at a maximum concentration of 20% does not significantly affect trypsin activity, and that SDC is precipitated by adding trifluoroacetic acid to a protein digestion reaction solution containing SDC. It describes that SDC can be removed by centrifugation prior to analysis. PT / JP2008 / 068124 Non-Patent Document 2 shows that the use of an acid-labile surfactant in a trypsin digestion buffer increases protein solubilization and degradation, and trypsin digestion. It describes that acid labile surfactant can be removed by centrifugation after acidifying the buffer.
非特許文献 1 Zhou et al. , Journal of Proteome Research 2006， 5，  Non-Patent Literature 1 Zhou et al., Journal of Proteome Research 2006, 5,
Analysis of Rat Hippocampal Plasma MembraneJ Analysis of Rat Hippocampal Plasma MembraneJ
非特許文献 2 Chen et al.， Journal of Proteome Research 2007 Jul ; 6 (7)： 2529 - 38 「0ptimization of Mass Spectrometry- Compatible Surfactants for Shotgun ProteomicsJ 発明の開示  Non-Patent Document 2 Chen et al., Journal of Proteome Research 2007 Jul; 6 (7): 2529-38 “0ptimization of Mass Spectrometry- Compatible Surfactants for Shotgun Proteomics
しかし、 上記のようなタンパク質試料の消化後に界面活性剤を沈殿除去する手 法によれば、界面活性剤による分析装置の汚染の問題を回避することはできるが、 タンパク質消化画分の回収効率は十分なものではない。 これは、 消化反応溶液中 に生じる界面活性剤の沈殿を固液分離する際に、 一部のタンパク質画分を欠失し てしまうことに起因するものと考えられる。  However, the method of precipitating and removing the surfactant after digestion of the protein sample as described above can avoid the problem of contamination of the analyzer by the surfactant, but the recovery efficiency of the protein digestion fraction is Not enough. This is thought to be due to the loss of some protein fractions during solid-liquid separation of the surfactant precipitate that occurs in the digestion reaction solution.
そこで本発明は、 界面活性剤による分析装置の汚染の問題を回避しつつ、 分析 対象タンパク質を十分に酵素消化することができ、 消化後のタンパク質又はぺプ チド断片を十分に回収することができる、 タンパク質試料の調製方法を提供する ことを目的とする。  Therefore, the present invention can sufficiently digest the protein to be analyzed while avoiding the problem of contamination of the analyzer due to the surfactant, and can sufficiently recover the digested protein or peptide fragment. An object of the present invention is to provide a method for preparing a protein sample.
本発明者らは、 界面活性剤を使用してタンパク質を可溶化及びプロテアーゼ消 化し、 その後、 界面活性剤を除去する手法において、 界面活性剤を脂溶化して有 機溶媒に分離することで、 タンパク質消化画分を効率的に回収できることを見出 し、 本発明を完成させるに至った。  In the method of solubilizing and protease-dissolving a protein using a surfactant, and then removing the surfactant, the surfactant is fat-solubilized and separated into an organic solvent. The inventors have found that a protein digested fraction can be efficiently recovered, and have completed the present invention.
すなわち、 本発明は以下の特徴を包含する。  That is, the present invention includes the following features.
( 1 ) (a) (i)分析対象タンパク質； （ii) l種又は 2種以上の脂溶性に改変可能な 界面活性剤；及び（ii i)プロテアーゼを含む反応溶液中で、分析対象タンパク質を 消化するステップと、 (b)該反応溶液に前記界面活性剤を脂溶化する試薬を添加するステップとを含 み、 (1) (a) (i) Protein to be analyzed; (ii) l type or two or more lipophilic surfactants; and (ii i) a protein to be analyzed in a reaction solution containing a protease. Digestion step, (b) adding a reagent for fat-solubilizing the surfactant to the reaction solution,
脂溶化された前記界面活性剤を有機溶媒中に分離することを特徴とする、 タンパ ク質試料の調製方法。 A method for preparing a protein sample, comprising separating the fat-solubilized surfactant in an organic solvent.
(2) 脂溶性に改変可能な界面活性剤がィオン性界面活性剤であることを特徴と する （1) 記載の方法。  (2) The method according to (1), wherein the surfactant capable of being modified to be fat-soluble is an ionic surfactant.
(3) イオン性界面活性剤はドデシル硫酸ナトリウム （SDS) であることを特徴と する （2) 記載の方法。  (3) The method according to (2), wherein the ionic surfactant is sodium dodecyl sulfate (SDS).
( 4 )前記界面活性剤を脂溶化する試薬は力リゥム塩であることを特徴とする（3) 記載の方法。  (4) The method according to (3), wherein the reagent for fat-solubilizing the surfactant is a strong Rium salt.
( 5 )イオン性界面活性剤はカルボン酸型界面活性剤であることを特徴とする（2) 記載の方法。  (5) The method according to (2), wherein the ionic surfactant is a carboxylic acid type surfactant.
(6) カルボン酸型界面活性剤はコール酸ナトリウム、 グリココール酸、 ラウリ ルサルコシン酸ナトリウム、 デォキシコール酸ナトリウム、 グリコケノデォキシ コール酸ナトリウム、 ケノデォキシコール酸ナトリゥム又はウルソデォキシコ一 ル酸ナトリウムであることを特徴とする （5) 記載の方法。  (6) Carboxylic acid type surfactants are sodium cholate, glycocholic acid, sodium lauryl sarcosinate, sodium deoxycholate, sodium glycochenodeoxycholate, sodium kenodeoxycholate or sodium ursodeoxycolate. (5) The method described in (5) above.
(7) 脂溶性に改変可能な界面活性剤が酸不安定界面活性剤であることを特徴と する （1) 記載の方法。  (7) The method according to (1), wherein the surfactant that can be modified to be lipophilic is an acid labile surfactant.
(8)酸不安定界面活性剤は、 3- [(2 -メチル- 2-ゥンデシル- 1， 3 -ジォキソラン -4- ィル）メ トキシル] -1-プロパンスルホン酸ナトリゥム又は 3-[3-(1, 1-アルキルォ キシェチル）ピリジン- 1-ィル]プロパン- 1-スルホン酸であることを特徴とする (8) Acid labile surfactants include 3-[(2-methyl-2-undecyl-1,3-dioxol-4-yl) methoxyl] -1-propanesulfonic acid sodium or 3- [3- It is characterized by being (1, 1-alkyloxychetyl) pyridine-1-yl] propane-1-sulfonic acid
(7) 記載の方法。 (7) The method described.
( 9)前記界面活性剤を脂溶化する試薬は酸であることを特徴とする（5)又は（7) 記載の方法。  (9) The method according to (5) or (7), wherein the reagent for fat-solubilizing the surfactant is an acid.
(10) 前記酸はトリフルォロ酢酸であることを特徴とする （9) 記載の方法。 (10) The method according to (9), wherein the acid is trifluoroacetic acid.
(1 1) 2種以上の脂溶性に改変可能な界面活性剤は、 カルボン酸型界面活性剤 から選択されることを特徴とする（1)記載の方法。 (1 1) The method according to (1), wherein the two or more types of surfactants that can be modified to be lipophilic are selected from carboxylic acid type surfactants.
(1 2) カルボン酸型界面活性剤は、 デォキシコール酸ナトリゥム及ぴラゥリル サルコシン酸ナトリゥムであることを特徴とする（11)記載の方法。 ( 1 3 ) 前記有機溶媒は予め前記反応溶液中に混合されていることを特徴とする (1)記載の方法。 (1 2) The method according to (11), wherein the carboxylic acid type surfactant is sodium deoxycholate and sodium lauryl sarcosinate. (1 3) The method according to (1), wherein the organic solvent is previously mixed in the reaction solution.
( 1 4 ) 前記反応溶液はマイクロエマルジヨンの形態であることを特徴とする (13)記載の方法。  (14) The method according to (13), wherein the reaction solution is in the form of microemulsion.
( 1 5 ) ステップ (b)後に前記有機溶媒を前記反応溶液に添加し、脂溶化された前 記界面活性剤を有機溶媒中に分離することを特徴とする（1)記載の方法。  (15) The method according to (1), wherein after step (b), the organic solvent is added to the reaction solution, and the fat-solubilized surfactant is separated into the organic solvent.
( 1 6 ) 前記有機溶媒はォクタノール、 酢酸ェチル、 ジェチルエーテル、 クロ口 ホルム又はへキサノールであることを特徴とする（1)記載の方法。  (16) The method according to (1), wherein the organic solvent is octanol, ethyl acetate, jetyl ether, chloroform, or hexanol.
( 1 7 ) 分析対象タンパク質は電気泳動による分離後に切り出したゲルに含まれ る目的のタンパク質であることを特徴とする（1)記載の方法。  (17) The method according to (1), wherein the protein to be analyzed is a target protein contained in a gel cut out after separation by electrophoresis.
( 1 8 ) (1)〜 Q7)のいずれか記載の方法によりタンパク質試料を調製し、分析に 供することを特徴とするタンパク質の分析方法。  (18) A protein analysis method comprising preparing a protein sample by the method according to any one of (1) to Q7) and subjecting the sample to analysis.
( 1 9 ) 前記分析は該サンプルを液体クロマトグラフィー質量分析（LC- MS) 又は マトリックス支援レーザー脱離ィオン化質量分析 （MALDI - MS) であることを特徴 とする（18)記載のタンパク質の分析方法。  (19) The analysis of the protein according to (18), wherein the analysis is liquid chromatography mass spectrometry (LC-MS) or matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS). Method.
本明細書は本願の優先権の基礎である日本国特許出願 2007- 259176号の明細書 および Zまたは図面に記載される内容を包含する。 図面の簡単な説明  This specification includes the contents described in the specification and Z or drawings of Japanese Patent Application No. 2007-259176, which is the basis of the priority of the present application. Brief Description of Drawings
図 1は、 SDC を用いた相間移動法、 酸沈殿法及び尿素法における大腸菌膜画分 のタンパク質同定結果の比較を示す。  Figure 1 shows a comparison of the protein identification results of the E. coli membrane fraction in the phase transfer method, acid precipitation method, and urea method using SDC.
図 2は、本発明において、界面活性剤として 10%ドデシル硫酸ナトリゥム（SDS) を、有機溶媒として消化反応溶液に対して 2倍用量のォクタノール（OctOH) を使 用した場合に、 脂溶化 SDSを完全に液液分離できたことを示す。  Fig. 2 shows that when 10% sodium dodecyl sulfate (SDS) is used as a surfactant and double dose of octanol (OctOH) is used as an organic solvent in the present invention, the fat-solubilized SDS is used. It shows that liquid-liquid separation was complete.
図 3は、 本発明において、 界面活性剤として 10%コール酸ナトリウムを、 有機 溶媒として消化反応溶液に対して 2倍用量のォクタノール（OctOH) を使用した場 合に、 脂溶化コール酸ナトリゥムを完全に液液分離できたことを示す。  Figure 3 shows the complete fat-solubilized sodium cholate in the present invention when 10% sodium cholate was used as the surfactant and doubled octanol (OctOH) was used as the organic solvent for the digestion reaction solution. Shows liquid-liquid separation.
図 4は、 本発明において、 界面活性剤として 10%グリココール酸を、 有機溶媒 として消化反応溶液と等量のォクタノール（OctOH) を使用した場合に、脂溶化グ リココール酸を完全に液液分離できたことを示す。 FIG. 4 shows that when 10% glycocholic acid is used as a surfactant and octanol (OctOH) is used as an organic solvent in an amount equal to that of a digestion reaction solution in the present invention. It shows that lycocholic acid was completely liquid-liquid separated.
図 5は、 本発明において、 界面活性剤として 1%ラウリルサルコシン酸ナトリ ゥムを、 有機溶媒として消化反応溶液に対して 0. 5倍用量のォクタノールを使用 した場合に、 脂溶化ラゥリルサルコシン酸ナトリゥムを完全に液液分離すること ができたことを示す。  FIG. 5 shows that when 1% sodium lauryl sarcosinate is used as a surfactant and 0.5 times the amount of octanol is used as an organic solvent in the present invention, the fat-solubilized lauryl sarcosine acid is used. This shows that sodium was completely liquid-liquid separated.
図 6は、 本発明において、 界面活性剤として 10%デォキシコール酸ナトリウム を、 有機溶媒として消化反応溶液に対して 0. 5倍用量のォクタノールを使用した 場合に、 脂溶化デォキシコール酸ナトリゥムを完全に液液分離することができた ことを示す。  FIG. 6 shows that in the present invention, when 10% sodium deoxycholate is used as a surfactant and 0.5 times the amount of octanol is used as an organic solvent with respect to the digestion reaction solution, the fat-solubilized sodium deoxycholate is completely liquid. Indicates that the liquid could be separated.
図 7は、 本発明において、 界面活性剤として 10%デォキシコール酸ナトリウム を、 有機溶媒として消化反応溶液と等量のへキサノールを使用した場合に、 脂溶 化デォキシコール酸ナトリゥムを完全に液液分離することができたことを示す。 図 8は、 本発明において、 界面活性剤として 1%デォキシコール酸ナトリウム を、 有機溶媒として消化反応溶液と等量の酢酸ェチルを使用した場合に、 脂溶化 デォキシコール酸ナトリゥムを完全に液液分離することができたことを示す。 図 9は、 本発明において、 界面活性剤として 1%デォキシコール酸ナトリウム を、 有機溶媒として消化反応溶液に対して 6倍用量のジェチルエーテルを使用し た場合に、 脂溶化デォキシコール酸ナトリゥムを完全に液液分離することができ たことを示す。  FIG. 7 shows that in the present invention, when 10% sodium deoxycholate is used as the surfactant and hexanol equivalent to the digestion reaction solution is used as the organic solvent, the fat-soluble sodium deoxycholate is completely liquid-liquid separated. Indicates that it was possible. FIG. 8 shows that in the present invention, when 1% sodium deoxycholate is used as a surfactant and ethyl acetate equivalent to the digestion reaction solution is used as an organic solvent, the fat-solubilized sodium deoxycholate is completely liquid-liquid separated. It shows that it was possible. FIG. 9 shows that in the present invention, 1% sodium deoxycholate was used as a surfactant, and 6 times the amount of jetyl ether was used as an organic solvent for the digestion reaction solution. Indicates that liquid-liquid separation was possible.
図 1◦は、 本発明において、 界面活性剤として 1%デォキシコール酸ナトリウ ムを、 有機溶媒として消化反応溶液に対して 8倍用量のクロ口ホルムを使用した 場合に、 脂溶化デォキシコール酸ナトリゥムを完全に液液分離することができた ことを示す。  Fig. 1◦ shows that in the present invention, 1% sodium deoxycholate was used as the surfactant, and 8 times the dose of black mouth form with respect to the digestion reaction solution was used as the organic solvent. Shows that liquid-liquid separation was possible.
図 1 1は、 本発明において、 界面活性剤として l%RapiGeSt™SF を、 有機溶媒 として消化反応溶液に対して 0. 5倍用量のオタタノールを使用した場合に、 脂溶 化 RapiGest™ SFを完全に液液分離することができたことを示す。 Figure 1 1, in the present invention, the l% RapiG eS t ™ SF as a surfactant, when using Otatanoru of 0.5 times the dose to digestion reaction solution as the organic solvent, Abura溶of RapiGest ™ SF Indicates that liquid-liquid separation could be completed.
図 1 2は、 本発明において、 界面活性剤として 1%グリコケノデォキシコール 酸ナトリゥムを、 有機溶媒として消化反応溶液に対して 0. 5倍用量のォクタノー ルを使用した場合に、 脂溶化ダリコケノデォキシコール酸ナトリゥムを完全に液 液分離することができたことを示す。 Fig. 12 shows the fat-solubilization in the present invention when 1% glycochemodeoxychol sodium is used as a surfactant and 0.5 times the amount of octanool as an organic solvent is used as a digestion reaction solution. Completely liquid sodium daricochenodeoxycholate This shows that liquid separation was possible.
図 1 3は、 本発明において、 界面活性剤として 10%ケノデォキシコール酸ナト リウムを、 有機溶媒として消化反応溶液に対して 1. 5倍用量のォクタノールを使 用した場合に、 脂溶化ケノデォキシコール酸ナトリゥムを完全に液液分離するこ とができたことを示す。  Fig. 13 shows the fat solubilization in the present invention when 10% sodium kenodeoxycholate was used as the surfactant and 1.5 times the amount of octanol was used as the organic solvent for the digestion reaction solution. This shows that sodium kenodeoxycholate was completely liquid-liquid separated.
図 1 4は、 本発明において、 界面活性剤として 10%ウルソデォキシコール酸ナ トリウムを、 有機溶媒として消化反応溶液に対して 3. 5倍用量のォクタノールを 使用した場合に、 脂溶化ウルソデォキシコール酸ナトリゥムを完全に液液分離す ることができたことを示す。  Fig. 14 shows fat-solubilized urso when 10% sodium ursodeoxycholate is used as a surfactant and 3.5 times the dose of octanol is used as an organic solvent in a digestion reaction solution. This shows that sodium deoxycholate was completely liquid-liquid separated.
図 1 5は、 SDC、 SLSおよび SDC- SLS混合系を用いた相間移動法による大腸菌膜 画分のタンパク質同定結果の比較を示す。 なお、 図中の数値は大腸菌膜画分から 同定されたタンパク質の総数を示し、 カツコ内の数値は同定された膜タンパク質 の数を示す。  Fig. 15 shows a comparison of protein identification results of E. coli membrane fractions by the phase transfer method using SDC, SLS and SDC-SLS mixed systems. The numbers in the figure indicate the total number of proteins identified from the E. coli membrane fraction, and the numbers in Katsuko indicate the number of membrane proteins identified.
図 1 6は、 SDC、 SLSおよび SDC- SLS混合系を用いた相間移動法による大腸菌膜 画分の同定膜タンパク質における膜貫通領域数の比較を示す。 発明を実施するための最良の形態  Figure 16 shows a comparison of the number of transmembrane domains in the identified membrane proteins of the E. coli membrane fraction by phase transfer using SDC, SLS and SDC-SLS mixed systems. BEST MODE FOR CARRYING OUT THE INVENTION
本発明のタンパク質試料の調製方法は、 タンパク質の可溶化に用いた界面活性 剤を脂溶性に改変することにより、該界面活性剤を有機溶媒中に分離する（以下.、 In the method for preparing a protein sample of the present invention, the surfactant used for solubilizing the protein is modified to be fat-soluble, thereby separating the surfactant into an organic solvent (hereinafter,...
「相間移動法」 とも称する） ことを特徴とする。 この方法によれば、 分析装置へ の影響が懸念される界面活性剤をプロテァーゼ処理後のタンパク質画分を含む試 料から除去することができる。 一方、 界面活性剤の沈殿形成によるタンパク質画 分の一部欠失の問題を回避することができるため、 タンパク質回収効率の向上を 可能にするという利点を有する。 It is also called “phase transfer method”. According to this method, it is possible to remove the surfactant, which is likely to affect the analyzer, from the sample containing the protein fraction after the protease treatment. On the other hand, the problem of partial deletion of protein fractions due to the formation of surfactant precipitates can be avoided, and this has the advantage of improving the protein recovery efficiency.
具体的に、 本発明の方法は、 （a) (i)分析対象タンパク質； （ii)脂溶性に改変可 能な界面活性剤；及び（iii)プロテアーゼを含む反応溶液中で、分析対象タンパク 質を消化するステップと、（b)該反応溶液に前記界面活性剤を脂溶化する試薬を添 加するステップとを包含する。  Specifically, the method of the present invention comprises: (a) (i) a protein to be analyzed; (ii) a surfactant that can be modified to be lipophilic; and (iii) a protein to be analyzed in a reaction solution containing a protease. And (b) adding a reagent for fat-solubilizing the surfactant to the reaction solution.
ステップ（a)は、脂溶性に改変可能な界面活性剤を含む溶液に分析対象タンパク 質を溶解し、 プロテァーゼ消化するステップである。 In step (a), the protein to be analyzed is added to a solution containing a surfactant that can be modified to be lipophilic. It is a step of dissolving the quality and digesting the protease.
分析対象のタンパク質は特に制限されず、 例えば、 組織、 生体液、 細胞、 細胞 器官等の破砕物、 タンパク質抽出物などを常法に従って調製して使用することが できる。 本発明では、 分析対象タンパク質が、 膜タンパク質、 GPIアンカー（ダリ コシルフォスファチジルイノシトールアンカー）化などの翻訳後修飾を受けたタ ンパク質、 膜付着タンパク質などの不溶性、 難溶性タンパク質を含む場合に特に 有効である。  The protein to be analyzed is not particularly limited, and for example, tissues, biological fluids, cells, pulverized organs, protein extracts and the like can be prepared and used according to conventional methods. In the present invention, the protein to be analyzed includes a membrane protein, a protein having undergone post-translational modification such as GPI anchor (daricosylphosphatidylinositol anchor), or an insoluble or hardly soluble protein such as a membrane-attached protein. It is particularly effective for
「脂溶性に改変可能な界面活性剤」 とは、特定の試薬の添加に起因する PHの変 化などにより、 その全体又は一部の脂溶性が増大する界面活性剤を指す。 本発明 において、 脂溶性に改変可能な界面活性剤として、 例えば、 イオン性界面活性剤 又は酸不安定界面活性剤を挙げることができる。  The “surfactant that can be modified to be lipophilic” refers to a surfactant that increases in whole or part of its lipophilicity due to, for example, a change in PH caused by the addition of a specific reagent. In the present invention, examples of the surfactant that can be modified to be lipophilic include an ionic surfactant and an acid labile surfactant.
本発明に使用することができるィォン性界面活性剤は、 陽ィォン界面活性剤及 び陰イオン界面活性剤の両者を含む。 本発明において、 イオン性界面活性剤とし て、 酸性条件下で脂溶性に改変されるカルボン酸型界面活性剤を用いることが好 ましい。 そのようなカルボン酸型界面活性剤として、 これに限定されるものでは ないが、 例えば、 コール酸ナトリウム、 デォキシコール酸ナトリウム、 グリココ ール酸、 グリコケノデォキシコール酸ナトリウム、 ケノデォキシコール酸ナトリ ゥム、 ゥルソデォキシコ一ル酸ナトリウムなどのコール酸類、 ラウリルサルコシ ン酸ナトリゥムなどのサルコシン類、 アミノ基をアルキル化したアミノ酸誘導体 などが挙げられる。 また本発明において、 イオン性界面活性剤として、 硫酸型界 面活性剤を使用することも同様に好ましい。そのような硫酸型界面活性剤として、 力リゥムを添加することにより脂溶性に改変可能なドデシル硫酸ナトリウム (SDS) などを挙げることができる。  The cationic surfactants that can be used in the present invention include both cationic surfactants and anionic surfactants. In the present invention, it is preferable to use a carboxylic acid type surfactant that is modified to be lipophilic under acidic conditions as the ionic surfactant. Examples of such carboxylic acid type surfactants include, but are not limited to, sodium cholate, sodium deoxycholate, glycocholic acid, sodium glycochenodeoxycholate, chenodeoxycol. Examples thereof include cholic acids such as sodium acid sodium and sodium sodium sodium hydroxysuccinate, sarcosines such as sodium lauryl sarcosinate, and amino acid derivatives obtained by alkylating amino groups. In the present invention, it is also preferable to use a sulfuric acid type surfactant as the ionic surfactant. Examples of such a sulfuric acid type surfactant include sodium dodecyl sulfate (SDS) which can be modified to be lipophilic by adding force lime.
本明細書中、 酸不安定界面活性剤は、 酸性条件下で分子内構造における疎水性 部が切り離されることにより脂溶性を獲得する界面活性剤を指し、 酸性条件下で 親水性部と疎水性部とに切断されるものであればイオン性であっても非ィォン性 であってもよい。 タンパク質可溶化能を考慮すれば、 イオン性の酸不安定界面活 性剤を使用することが好ましい。このような酸不安定界面活性剤としては、 3- [ (2 - メチル- 2-ゥンデシル- 1, 3 -ジォキソラン- 4 -ィノレ）メ トキシル] -1-プロパンスルホ ン酸ナトリゥム、 3_ [3- (1， 1 -アルキルォキシェチル)ピリジン- 1-ィル]プロパン -1 -スルホン酸などが挙げられ、それぞれ商品名 Rapigest™SF (Water Corporation, Milford, MA)、 PPS SILENT™ (Protein Discovery, Inc. , Knoxville, TN) とし て市販されている。 In this specification, an acid labile surfactant refers to a surfactant that acquires lipophilicity by separation of the hydrophobic part in the intramolecular structure under acidic conditions. The hydrophilic part and hydrophobicity under acidic conditions It may be ionic or nonionic as long as it can be cut into parts. In view of protein solubilization ability, it is preferable to use an ionic acid labile surfactant. Such acid labile surfactants include 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-inole) methoxyl] -1-propanesulfone Sodium acetate, 3_ [3- (1,1-alkyloxetyl) pyridine-1-yl] propane-1-sulfonic acid, etc., and trade names Rapigest ™ SF (Water Corporation, Milford, MA ), PPS SILENT ™ (Protein Discovery, Inc., Knoxville, TN).
以下に Rapigest™ SF、 PPS SILENT™の構造を記載する ：  The structure of Rapigest ™ SF and PPS SILENT ™ is described below:
化 1  1
Figure imgf000010_0001
化 2
Figure imgf000010_0001
2
Figure imgf000010_0002
本発明では、 酸不安定界面活性剤として、 上記市販の酸不安定界面活性剤を使 用することができる。
Figure imgf000010_0002
In the present invention, the above-mentioned commercially available acid labile surfactant can be used as the acid labile surfactant.
その他、 ステップ （a) で使用することができる界面活性剤として、 双性界面活 性剤を挙げることができる。 双性界面活性剤は、 水に溶解した際、 アルカリ性領 域では陰ィオン界面活性剤の性質を、 酸性領域では陽ィオン界面活性剤の性質を 示す界面活性剤であり、 非限定的に CHAPS、 3- [ (3 -コラミ ドプロピル）ジメチルァ ンモ -ォ] - 2 -ヒ ドロキシプロパンスルホン酸（CHAPS0)、 アルキルべタイン類など を挙げることができる。  In addition, examples of the surfactant that can be used in step (a) include a zwitterionic surfactant. A zwitterionic surfactant is a surfactant that, when dissolved in water, exhibits the properties of an anionic surfactant in the alkaline region and the properties of a cationic surfactant in the acidic region, including but not limited to CHAPS, Examples include 3-[(3-choramidopropyl) dimethylan-o] -2-hydroxypropanesulfonic acid (CHAPS0), alkylbetaines, and the like.
プロテアーゼ消化反応溶液中に添加する上記界面活性剤の濃度は、 使用する界 面活性剤に応じて異なるが、 プロテアーゼを不活化しない濃度であれば特に限定 されない。 したがって、 従来使用されてきた界面活性剤の濃度に比較して、 より 高い濃度で使用することが可能であるという利点を有する。 すなわち、 本発明の 相間移動法は、 従来技術で認められる界面活性剤の沈殿形成に起因するタンパク 質画分の一部欠失の問題を生じないため、 より高濃度の界面活性剤の添加が可能 であり、 その結果、 従来同定することができなかった疎水性の高いタンパク質を も可溶化することができる。 これにより、 疎水性の高いタンパク質に対してもプ 口テアーゼによる消化が可能になる。 The concentration of the surfactant added to the protease digestion reaction solution depends on the field of use. Although it varies depending on the surfactant, it is not particularly limited as long as it does not inactivate the protease. Therefore, it has the advantage that it can be used at a higher concentration than the concentration of the surfactant that has been conventionally used. That is, the phase transfer method of the present invention does not cause the problem of partial deletion of the protein fraction caused by the precipitate formation of the surfactant recognized in the prior art. As a result, it is possible to solubilize highly hydrophobic proteins that could not be identified in the past. This makes it possible to digest even highly hydrophobic proteins with protease.
例えば、 上記界面活性剤の添加濃度は、 使用する界面活性剤に応じて、 0. 01〜 20% (w/v)、 好ましくは 0. 01〜15% (w/v)、 より好ましくは 0. 01〜10% (w/v)、 例えば 1% (w/v) とすることができる。 当業者は使用する界面活性剤の種類に応 じて、 プロテアーゼを不活化しない最適な界面活性剤の濃度を適宜設定すること ができる。  For example, the concentration of the surfactant added is 0.01 to 20% (w / v), preferably 0.01 to 15% (w / v), more preferably 0, depending on the surfactant used. 01-10% (w / v), for example 1% (w / v). A person skilled in the art can appropriately set the optimum surfactant concentration that does not inactivate the protease according to the type of surfactant used.
上記界面活性剤の濃度は、 脂溶化後に消化反応溶液の 0. 5〜20倍用量、 好まし くは 0. 5〜15倍用量、 より好ましくは 0. 5〜： 10倍用量の有機溶媒に溶解する濃度 であることが好ましい。  The concentration of the surfactant is 0.5 to 20 times the amount of the digestion reaction solution after fat solubilization, preferably 0.5 to 15 times the dose, more preferably 0.5 to 10 times the amount of the organic solvent. The concentration is preferably dissolved.
ステップ（a)で使用するプロテアーゼは特に制限されず、タンパク質消化の目的 に応じて当業者はプロテアーゼを適宜選択することができる。 例えば、 本発明の 方法により調製したタンパク質試料を質量分析に使用する場合には、 プロテア一 ゼとしてトリプシンを使用することが好ましい。 なお、 酵素処理条件として特別 な方法を用いる必要はなく、 当業者は常法に従って処理温度、 pH、 酵素添加量を 設定することができる。  The protease used in step (a) is not particularly limited, and those skilled in the art can appropriately select a protease according to the purpose of protein digestion. For example, when a protein sample prepared by the method of the present invention is used for mass spectrometry, trypsin is preferably used as a protease. In addition, it is not necessary to use a special method as the enzyme treatment conditions, and those skilled in the art can set the treatment temperature, pH, and amount of enzyme added according to conventional methods.
ステップ (b)は、 ステツプ（a)で使用した界面活性剤を脂溶化するステップであ る。 具体的にステップ（b)では、 プロテアーゼ消化反応溶液に、 ステップ（a)で使 用した界面活性剤を脂溶化する試薬を添加する。 添加すべき試薬はステップ（a) で使用した界面活性剤に応じて異なるが、 典型的には酸又はカリゥム塩である。 ステップ (b)で使用することができる酸の形態は特に制限されるものではなく、 例えばトリフルォロ酢酸 （TFA)、 塩酸、 硫酸、 リン酸、 へキサフルォロ酪酸、 酢 酸、 ギ酸などを挙げることができる。 またその添加量は、 プロテアーゼ消化反応 溶液の pHを酸性、 すなわち pH0〜4、 好ましくは pH0〜3にするのに十分な量であ る。 上記添加量の酸により、 プロテアーゼ消化反応溶液中の界面活性剤の少なく とも 50%、 好ましくは少なくとも 70%、 より好ましくは少なくとも 80%、 より 好ましくは少なくとも 90%、そして最も好ましくは少なくとも 95%が脂溶性に改 変されることが好ましい。 Step (b) is a step of solubilizing the surfactant used in step (a). Specifically, in step (b), a reagent for fat-solubilizing the surfactant used in step (a) is added to the protease digestion reaction solution. The reagent to be added depends on the surfactant used in step (a), but is typically an acid or potassium salt. The form of the acid that can be used in step (b) is not particularly limited, and examples thereof include trifluoroacetic acid (TFA), hydrochloric acid, sulfuric acid, phosphoric acid, hexafluorobutyric acid, acetic acid, formic acid and the like. . The amount added is the protease digestion reaction It is an amount sufficient to make the pH of the solution acidic, that is, pH 0-4, preferably pH 0-3. Depending on the amount of acid added, at least 50%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the surfactant in the protease digestion reaction solution. It is preferable to change to fat solubility.
ステップ（b)で使用することができるカリウム塩の形態も特に制限されなレ、。例 えば塩化カリウム、 リン酸二水素カリウム、 酢酸カリウム、 ギ酸カリウムなど、 任意のカリウム塩の形態を使用することができる。 またその添加量は、 プロテア ーゼ消化反応溶液に含まれる界面活性剤 （例えば SDSなど） の濃度に応じて異な るが、 該反応溶液中に含まれる界面活性剤の少なくとも 50%、 好ましくは少なく とも 70%、 より好ましくは少なくとも 80%、 より好ましくは少なくとも 90%、 そして最も好ましくは少なくとも 95%が脂溶性に改変される量に設定すること が好ましい。  The form of the potassium salt that can be used in step (b) is also not particularly limited. For example, any potassium salt form such as potassium chloride, potassium dihydrogen phosphate, potassium acetate, potassium formate can be used. The amount of addition varies depending on the concentration of the surfactant (eg, SDS) contained in the protease digestion reaction solution, but is at least 50%, preferably less than the surfactant contained in the reaction solution. It is preferred to set the amount to 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95% modified to be fat-soluble.
このように、ステップ（a)で使用した界面活性剤を脂溶化することにより、界面 活性剤を有機溶媒中に分離することができる。  Thus, the surfactant can be separated into the organic solvent by solubilizing the surfactant used in step (a).
有機溶媒は、 予めプロテアーゼ消化反応溶液中に添加されていてもよいし、 界 面活性剤を脂溶化する試薬と同時に、 又は該試薬の添加後に添加してもよい。 有機溶媒を予めプロテアーゼ消化反応溶液中に添加する場合には、 該溶液をェ マルジヨンの形態とすることが好ましい。 ェマルジヨンとは、 油滴が水中に分散 している状態を指し、 この状態では水と接触する油滴の表面積が増大している。 したがって、 タンパク質をより溶解し易くするという利点を有する。 ェマルジョ ンは、 水と有機溶媒とを含む溶液を激しく振とうすることにより容易に作製する ことができる。  The organic solvent may be added in advance to the protease digestion reaction solution, or may be added simultaneously with or after the addition of the reagent that solubilizes the surfactant. When an organic solvent is added in advance to the protease digestion reaction solution, the solution is preferably in the form of an emulsion. Emulsion refers to a state where oil droplets are dispersed in water, and in this state, the surface area of the oil droplets in contact with water is increased. Therefore, it has the advantage of making the protein easier to dissolve. An emulsion can be easily prepared by vigorously shaking a solution containing water and an organic solvent.
プロテアーゼ消化反応溶液はマイクロエマルジョンの形態を採ることがさらに 好ましい。 本明細書において、 マイクロェマルジョンは、 ェマルジヨン开態の中 でも特に油滴の粒径が約 10 i mとなるものを指す。 マイクロエマルジョンは通常 のェマルジョンに比較して水と接触する油滴の表面積がより大きくなり、 タンパ ク質の溶解性をより増大させることができるという利点を有する。 また、 消化反 応溶液がマイクロエマルジョンの形態を揉る際、 プロテアーゼは影響を受けない ことが確認されており、 プロテアーゼ活性の維持の点にも優れている。 さらに、 マイクロエマルジョンは通常のェマルジョンとは異なり、 静止状態でも数日間そ の形態を維持することができるという利点も有する。 More preferably, the protease digestion reaction solution takes the form of a microemulsion. In this specification, “microemulsion” refers to those in which the particle size of oil droplets is about 10 im, even among the emulsion openings. Microemulsions have the advantage that the surface area of the oil droplets in contact with water is larger than that of normal emulsion, and the solubility of the protein can be further increased. In addition, proteases are not affected when the digestion reaction solution is in the form of a microemulsion. It has been confirmed that it is excellent in maintaining protease activity. Furthermore, unlike ordinary emulsions, microemulsions have the advantage that they can remain in their form for several days even when they are stationary.
マイクロエマルジヨンは、 プロテアーゼ消化反応溶液中の水、 有機溶媒、 界面 活性剤、 及び場合により補助界面活性剤を所定の範囲の量にて、 混合し、 超音波 処理などを行うことにより作製できる。 詳細については、 例えば H. Kunieda and K Ohyama, J Colloid Interface Sci. , 1990, 136 (2) , 432-439を参照されたい。 有機溶媒を、 界面活性剤を脂溶化する試薬と同時に、 又は該試薬の添加後に添 加する場合には、 プロテアーゼ処理後に有機溶媒を添加することになるため、 有 機溶媒によるプロテアーゼの不活化を考慮する必要がないという利点がある。 本発明の方法で使用することができる有機溶媒は水と 2相に分離するものであ れば特に制限されるものではなく、 例えばォクタノール、 酢酸ェチル、 ジェチル エーテノレ、 クロロホノレム、 へキサノーノレ、 ヘプタノ一ノレ、 ジクロロメタン、 ジォ キサン、 ベンゼン、 トルエン、 へキサン、 ヘプタン、 オクタン、 ピリジンなどを 挙げることができる。  Microemulsions can be produced by mixing water, an organic solvent, a surfactant, and optionally an auxiliary surfactant in a predetermined range in a protease digestion reaction solution, and performing ultrasonic treatment or the like. For details, see, for example, H. Kunieda and K Ohyama, J Colloid Interface Sci., 1990, 136 (2), 432-439. When an organic solvent is added at the same time as the reagent that solubilizes the surfactant, or after the addition of the reagent, the organic solvent is added after the protease treatment. There is an advantage that there is no need to consider. The organic solvent that can be used in the method of the present invention is not particularly limited as long as it can be separated into water and two phases. For example, octanol, ethyl acetate, jetyl etherol, chlorohonolem, hexanol, heptanol monore , Dichloromethane, dioxane, benzene, toluene, hexane, heptane, octane, pyridine and the like.
また添加する有機溶媒の量は、 使用する界面活性剤の種類及び濃度、 並びに使 用する有機溶媒の種類に応じて、 当業者は適宜最適な添加量を設定することがで きる。 有機溶媒は、 脂溶化した界面活性剤の少なくとも 50%、 より好ましくは少 なくとも 70%、より好ましくは少なくとも 80%、より好ましくは少なくとも 90%、 そして最も好ましくは少なくとも 95%を溶解する量で添加することが好ましい。 本発明において、 2種以上の脂溶性に改変可能な界面活性剤を併用することも 可能である。 2種以上の界面活性剤の使用により、 単一の界面活性剤を使用する よりも低い界面活性剤の添加濃度でさえ、 プロテアーゼ処理後のタンパク質消化 画分の回収効率をさらに改善することができ、 より多くのタンパク質及ぴ膜タン パク質を同定することができるという利点がある。  Further, the amount of the organic solvent to be added can be appropriately determined by those skilled in the art depending on the type and concentration of the surfactant to be used and the type of the organic solvent to be used. The organic solvent is in an amount that dissolves at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the fat-solubilized surfactant. It is preferable to add. In the present invention, two or more kinds of surfactants that can be modified to be lipophilic can be used in combination. The use of two or more surfactants can further improve the recovery efficiency of protein digested fractions after protease treatment even at lower surfactant concentrations than using a single surfactant. There is an advantage that more proteins and membrane proteins can be identified.
2種以上の界面活性剤は、 同一の界面活性剤の群から選択してもよいし、 異な る界面活性剤の群から選択してもよい。 したがって、 2種以上の界面活性剤は、 イオン性界面活性剤、 酸不安定界面活性剤及ぴ双性界面活性剤よりなる群から選 択することができる。 好ましくは、 2種以上の界面活性剤は、同一の界面活性剤の群から選択される。 上記のとおり、 界面活性剤を脂溶化するために使用される試薬は、 使用する界面 活性剤の性質によって異なるため、 同一の界面活性剤の群に属する 2種以上の界 面活性剤を使用することにより、 複数の試薬を添加する煩雑さを回避することが できるからである。 例えば、 2種以上の界面活性剤が、 カルボン酸型界面活性剤 から選択される 2種以上、 又は酸不安定界面活性剤から選択される 2種以上であ る場合には、 これらの界面活性剤を脂溶化するために酸を添加するだけでよい。 同様に、 2種以上の界面活性剤が酸性条件下で脂溶性に改変されるカルボン酸 型界面活性剤と酸不安定界面活性剤との組合せであることも好ましい。 The two or more surfactants may be selected from the same surfactant group or may be selected from different surfactant groups. Therefore, the two or more surfactants can be selected from the group consisting of ionic surfactants, acid labile surfactants and zwitterionic surfactants. Preferably, the two or more surfactants are selected from the same group of surfactants. As described above, since the reagent used for fat-solubilizing the surfactant differs depending on the nature of the surfactant used, two or more surfactants belonging to the same surfactant group are used. This is because the complexity of adding a plurality of reagents can be avoided. For example, when two or more surfactants are two or more selected from carboxylic acid type surfactants or two or more selected from acid labile surfactants, these surfactants It is only necessary to add an acid in order to fat-solubilize the agent. Similarly, it is also preferred that the two or more surfactants are a combination of a carboxylic acid type surfactant and an acid labile surfactant that are modified to be lipophilic under acidic conditions.
特に好ましい界面活性剤の組合せは、 デォキシコール酸ナトリウムとラウリル サルコシン酸ナトリゥムの組合せである。  A particularly preferred surfactant combination is a combination of sodium deoxycholate and sodium lauryl sarcosinate.
本発明の方法は、ゲル内のタンパク質の消化に応用することができる。例えば、 電気泳動による分離後に切り出したゲルに含まれるタンパク質を消化、 抽出する 過程で本発明の方法を使用することができる。 具体的にはゲル内消化で用いるプ 口テアーゼ溶液に界面活性剤を共存させておき、 ゲル内消化終了後の生成したぺ プチドのゲルからの抽出過程においても抽出溶液の p H を中性、 またはアルカリ 性で行った後に、 ペプチドと界面活性剤を含む溶液に対し、 溶液消化時と同じく 有機溶媒を添加し、 溶液を酸性側にシフトさせることで、 ぺプチドを高い効率で 回収できる。 採用される電気泳動は、 電気泳動によりゲルを介してタンパク質が 分子量ゃ等電点の違いにより分離できる限り特に限定されず、 例えば SDS- PAGE、 等電点電気泳動、 2 次元電気泳動などを挙げることができる。 これにより、 比較 的発現量が低く、 従来法では同定が困難であったタンパク質の同定効率を向上さ せることができる。  The method of the present invention can be applied to the digestion of proteins in gels. For example, the method of the present invention can be used in the process of digesting and extracting a protein contained in a gel cut out after separation by electrophoresis. Specifically, a surfactant is allowed to coexist in the protease solution used for in-gel digestion, and the pH of the extracted solution is neutralized during the extraction process from the gel after completion of in-gel digestion. Alternatively, the peptide can be recovered with high efficiency by adding an organic solvent to the solution containing the peptide and the surfactant after the alkaline treatment, and shifting the solution to the acidic side as in the case of solution digestion. The electrophoresis used is not particularly limited as long as the protein can be separated by electrophoresis through a gel and the molecular weight is different due to the difference in isoelectric point. Examples include SDS-PAGE, isoelectric focusing, and two-dimensional electrophoresis. be able to. As a result, the protein expression efficiency, which is relatively low in expression level and difficult to identify by conventional methods, can be improved.
以上のように、 本発明の方法によれば、 膜タンパク質等の疎水性の高いタンパ ク質であっても十分に可溶化してプロテアーゼ処理を行うことができ、 また、 界 面活性剤を分離したとしても消化後のタンパク質画分を高効率に回収することが できる。 換言すれば、 本発明の方法によれば、 界面活性剤を分離するに際してプ 口テアーゼ処理後のタンパク質画分の一部が界面活性剤とともに欠失してしまう ような不都合を回避することができる。 したがって、 本発明の方法によれば、 例 えば質量分析装置等の各種分析装置を汚染する界面活性剤を十分に除去しながら も分析対象のタンパク質を確実に分析できるタンパク質試料を調製することがで きる。 As described above, according to the method of the present invention, even a highly hydrophobic protein such as a membrane protein can be sufficiently solubilized and treated with protease, and the surfactant is separated. Even so, the protein fraction after digestion can be recovered with high efficiency. In other words, according to the method of the present invention, it is possible to avoid the disadvantage that a part of the protein fraction after the protease treatment is lost together with the surfactant when separating the surfactant. . Thus, according to the method of the present invention, an example For example, it is possible to prepare a protein sample that can reliably analyze a protein to be analyzed while sufficiently removing a surfactant that contaminates various analyzers such as a mass spectrometer.
よって、本発明の方法により調製されたタンパク質試料は、その後、質量分析、 アミノ酸分析、 抗体を用いたィムノアッセィ等、 任意の分析に供することができ る。 例えば、 上記タンパク質試料を質量分析に供した場合、 従来の方法では同定 できなかったタンパク質画分を検出し、 同定することができる。 すなわち、 本発 明の方法によれば、質量分析等の分析系によってタンパク質画分を同定する際に、 従来の方法と比較して同定効率を大幅に向上させることができる。  Therefore, the protein sample prepared by the method of the present invention can be subjected to any analysis such as mass spectrometry, amino acid analysis, and immunoassay using an antibody. For example, when the protein sample is subjected to mass spectrometry, a protein fraction that could not be identified by a conventional method can be detected and identified. That is, according to the method of the present invention, when a protein fraction is identified by an analysis system such as mass spectrometry, the identification efficiency can be greatly improved as compared with the conventional method.
本発明の方法により調製したタンパク質試料を、 液体クロマトグラフィー質量 分析 （LC- MS) 又はマトリクス支援レーザー脱離イオン化質量分析 （MALDI- MS) に 供することが特に有効である。  It is particularly effective to subject the protein sample prepared by the method of the present invention to liquid chromatography mass spectrometry (LC-MS) or matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS).
以下、 実施例により本発明をより詳細に説明するが、 本発明はこれに限定され ない。  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to this.
実施例 1 ：本発明の相間移動法、 酸沈殿法及び尿素を用いた溶液消化法における タンパク質同定結果の比較 Example 1: Comparison of protein identification results in the phase transfer method, acid precipitation method and solution digestion method using urea of the present invention
試料調製 Sample preparation
本実施例では不溶性タンパク質の可溶化及び分解効率を調査するために不溶性 タンパク質としてタンパク質膜画分を用いた。  In this example, a protein membrane fraction was used as an insoluble protein in order to investigate the solubilization and degradation efficiency of the insoluble protein.
膜画分は大腸菌 K- 12株 BW25113から採取した。 BW25113は 50mLの LB培地で培 養 （37°C、 8時間） したものを 10分間遠心分離 （5000回転、 4°C) をして集菌し た。 集菌した BW25113は 5mLの 1M塩化力リウム、 10mM トリス溶液（洗浄バッフ ァー）で懸濁し、 10分間遠心分離 （5000回転、 4°C) して洗浄した。 洗浄作業は 2 回行った。 5mL の洗浄バッファーに懸濁した BW25113 を超音波破砕機 （T0MY, The membrane fraction was collected from E. coli K-12 strain BW25113. BW25113 was cultured in 50 mL of LB medium (37 ° C, 8 hours) and collected by centrifugation for 10 minutes (5000 rpm, 4 ° C). The collected BW25113 was suspended in 5 mL of 1M potassium chloride and 10 mM Tris solution (washing buffer), and washed by centrifugation (5000 rpm, 4 ° C) for 10 minutes. Washing was performed twice. BW25113 suspended in 5 mL of washing buffer was sonicated with an ultrasonic crusher (T0MY,
UD-201) で破砕し、 2500回転で 5分間、 遠心分離して沈殿物を除去した。 上清は 超遠心分離機（BECKMAN COULTER , Optima XL- 100K Ultracentrifuge)を用いてUD-201) and centrifuged at 2500 rpm for 5 minutes to remove the precipitate. The supernatant was obtained using an ultracentrifuge (BECKMAN COULTER, Optima XL-100K Ultracentrifuge).
40， 000回転で 1時間遠心分離し、遠心分離後の沈殿物を粗膜画分として回収した。 粗膜画分を 10mLの 0. 1M 炭酸ナトリゥム溶液に懸濁し、 500 // Lずつ 1. 5mLチュー ブに分注し、 15， 000回転で 10分間遠心分離した沈殿物を膜画分として回収した。 P T/JP2008/068124 膜画分は使用するまで- 30°Cで保存した。 Centrifugation was performed at 40,000 rpm for 1 hour, and the precipitate after centrifugation was recovered as a crude membrane fraction. Suspend the crude membrane fraction in 10 mL of 0.1 M sodium carbonate solution, dispense 500 // L at a time into a 1.5 mL tube, and collect the precipitate as a membrane fraction after centrifugation at 15,000 rpm for 10 minutes. did. PT / JP2008 / 068124 Membrane fractions were stored at -30 ° C until use.
膜タンパク質の可溶化剤には 0. 5%デォキシコール酸ナトリゥム（D0JIND0 Cat. No. D520 : SDC)及ぴ 8M尿素（和光純薬 Cat. No. 217-00615)を用いた。 採取した BW25113の膜画分に対して 500 // Lの可溶化剤を加えて懸濁した後、 95°Cで 5分間 加熱した。 膜タンパク質懸濁液は超音波発生器（T0CH0、 UC- 1331N)で 10分間処理 し、 不溶物は 15, 000回転で 10分間遠心分離して除いた。 50 /i Lの試料を用い、 5 /z Lの 10mMのジチオスレィ トール （和光純薬 Cat. No. 040-29223： DTT) を 5 L 加え、室温で 30分ィンキュベーションしてタンパク質中のシスティン残基を還元 した。 その後、 55mMのョードアセトアミ ド （和光純薬 Cat. No. 091-02153) を 5 L加え、 30分ィンキュベーションしてシスティン残基をアルキル化した。 以下 1)〜2) に示すように、 それぞれの可溶化剤でタンパク質を可溶化し、 その後プロ テアーゼで消化した。  As membrane protein solubilizers, 0.5% sodium deoxycholate (D0JIND0 Cat. No. D520: SDC) and 8M urea (Wako Pure Chemicals Cat. No. 217-00615) were used. To the collected membrane fraction of BW25113, 500 // L solubilizer was added and suspended, and then heated at 95 ° C for 5 minutes. The membrane protein suspension was treated with an ultrasonic generator (T0CH0, UC-1331N) for 10 minutes, and the insoluble matter was removed by centrifugation at 15,000 rpm for 10 minutes. Using 50 / i L of sample, add 5 L of 5 / z L of 10 mM dithiothreitol (Wako Pure Chemicals Cat. No. 040-29223: DTT), and incubate at room temperature for 30 minutes. The cysteine residue was reduced. Then, 5 L of 55 mM odoacetamide (Wako Pure Chemicals Cat. No. 091-02153) was added and incubated for 30 minutes to alkylate cysteine residues. As shown in 1) to 2) below, the protein was solubilized with each solubilizer and then digested with protease.
1) 8M尿素を用いた消化  1) Digestion with 8M urea
8M尿素で可溶化した上記のタンパク質試料溶液に、 0. 2mg/mLの Lys- C (和光純 薬 Cat No. 125- 05061)を 1 L加え、 室温で 4時間インキュベーションしタンパ ク質を消化した。 150 μ ί の 50mM 炭酸水素アンモ-ゥム緩衝液を加えた後、 0. 5mg/mLのトリプシン （プロメガ社製、 Cat. No. V511C) を 1 /i L力!]え、 室温で 24時間インキュベーションし、 タンパク質を消化した。 消化後、 トリフルォロ酢 酸（TFA)を終濃度で 1%となるように加え、 トリプシンを失活させた。 消化後の溶 液を以後の操作に用いた。  1 L of 0.2 mg / mL Lys-C (Wako Pure Chemicals Cat No. 125-05061) was added to the above protein sample solution solubilized with 8 M urea, and the protein was digested by incubation at room temperature for 4 hours. . After adding 150 μί of 50 mM ammonium bicarbonate buffer, add 0.5 mg / mL trypsin (Promega, Cat. No. V511C) to 1 / i L force!] For 24 hours at room temperature Incubate and digest protein. After digestion, trifluoroacetic acid (TFA) was added to a final concentration of 1% to inactivate trypsin. The digested solution was used for subsequent operations.
2) 0. 5%SDCを用いた消化  2) Digestion with 0.5% SDC
0. 5%SDC で可溶化した上記のタンパク質試料溶液は、 0. 5mg/mL のトリプシン (プロメガ社製、 Cat. No. V511C) を 加え、 室温で 24時間インキュベーシ ヨンして消化した。 SDC は酸沈殿法又は相間移動法で除去した。 酸沈殿法は消化 後の試料に TFAを終濃度で 1%となるように加え、不溶化した SDCを 13， 000回転 で 2分間、遠心分離して除去した。相間移動法は試料と等量の酢酸ェチル（和光純 薬 Cat. No. 055 - 05991)を加え、 TFAを終濃度で 1%になるように添加した。 2液 を懸濁させた後、 13, 000回転で 2分間遠心分離して、 2層に分配した。 SDCを含 む酢酸ェチル相（上層）をピぺッティングで除去した。 水相 (下層)は減圧乾燥機 2008/068124 The above protein sample solution solubilized with 0.5% SDC was digested by adding 0.5 mg / mL trypsin (Promega, Cat. No. V511C) and incubating at room temperature for 24 hours. SDC was removed by acid precipitation or phase transfer. In the acid precipitation method, TFA was added to the digested sample to a final concentration of 1%, and insoluble SDC was removed by centrifugation at 13,000 rpm for 2 minutes. In the phase transfer method, an equal amount of ethyl acetate (Wako Pure Chemicals Cat. No. 055-05991) was added to the sample, and TFA was added to a final concentration of 1%. After the two liquids were suspended, the mixture was centrifuged at 13,000 rpm for 2 minutes and distributed into two layers. The ethyl acetate phase (upper layer) containing SDC was removed by pipetting. The aqueous phase (lower layer) is a vacuum dryer. 2008/068124
(TOMY CC- 105)で 45分間処理した後、 以後の操作に用いた。 After treatment with (TOMY CC-105) for 45 minutes, it was used for subsequent operations.
LC-MS測定 LC-MS measurement
上記 1) ~ 2)の処理により調製した試料溶液について、以下のように LC- MS測定 を行った。  The sample solution prepared by the treatments 1) and 2) was subjected to LC-MS measurement as follows.
200 L用ピぺットチップと Empore C18ディスクを用いて C18-StageTip (自家製、 J. Rappsilber, Y. Ishihama, M. Mann, Anal Chem 75 (2003) 663)を作製した。 StageTipは 20 Lのメタノール、 20 μ Lの 80%ァセトニトリル、 0. 1%TFA (以下、 「溶液 という）及び の 5%ァセトニトリル、 0. 1%TFA (以下、 「溶液 A」 という）をロードして活性化させた。 活性化させた StageTipに、 上記 1)〜2)の処 理により調製した各試料溶液を StageTipにロードした。 20 μ Lの溶液 Αで洗浄し た後、 40 Lの溶液 Bで StageTipからペプチドを溶出させた。 溶出したペプチド 溶液は遠心濃縮した後、 20 μ Lの溶液 Αで溶解し、 LC- MS用試料溶液とした。  C18-StageTip (homemade, J. Rappsilber, Y. Ishihama, M. Mann, Anal Chem 75 (2003) 663) was prepared using a 200 L pipette tip and an Empore C18 disk. StageTip is loaded with 20 L of methanol, 20 μL of 80% acetonitrile, 0.1% TFA (hereinafter referred to as “solution”) and 5% acetonitrile, 0.1% TFA (hereinafter referred to as “solution A”). Activated. Each sample solution prepared by the above processes 1) to 2) was loaded onto StageTip. After washing with 20 μL of solution, the peptide was eluted from StageTip with 40 L of solution B. The eluted peptide solution was concentrated by centrifugation and then dissolved in 20 μL of solution to prepare a sample solution for LC-MS.
この試料溶液について LC (C18 column) /MS (ABI/Qstar XL) システムを用 いての測定を行った。 HPLC条件として、 C18シリ力ゲル（ReproSil- Pur 120 C18-AQ, The sample solution was measured using an LC (C18 column) / MS (ABI / Qstar XL) system. HPLC conditions include C18 Siri-Force Gel (ReproSil-Pur 120 C18-AQ,
3 t tn) を充填した自家製のエレク トロスプレ 体型カラム （Y. Ishihama, J.3 t tn) packed in homemade electrospray column (Y. Ishihama, J.
Rappsilber, J. S. Andersen, M. Mann, J Chromatogr A 979 (2002) 233. ) 0. 1x150mm に移動相 Aとして 0. 5%酢酸水、移動相 Bとして 80%ァセトニトリルを含む 0. 5% 酢酸水を用いて、初期 B濃度を 5%として、 最初の 15分間で直線的に 30%、 その 後 5分間で直線的に 100%とし、その後移動相 Bを 100%にして 5分間維持、その 後移動相 Bを 5%として 35分後に次のサンプルを注入した。 送液には DI0NEX社 の Ultimate 3000を用い、 500nL/分の流速で分析を行った。 試料溶液を CTC社の ォートサンプラー PALによって 5 μい注入し、試料を一度ィンジヱクタ一のサンプ ルループに注入した後に分析力ラムに送り込んだ。 日京テクノス社製 LC - MSィン ターフェースを装備した ABI社の Qstar XLを用いて、 ESI電圧として 2. 4kVを印 カロした。 測定は、 Information dependent acquisitionモードで、 1秒間の Survey スキャンの後、 最大 3つの MSMSスキャン （各 0. 6秒） を行った。 MSMSモード力 ら Surveyスキャンへのスィツチは 1スぺク トルとした。 Rappsilber, JS Andersen, M. Mann, J Chromatogr A 979 (2002) 233.) 0.1x150mm containing 0.5% acetic acid water as mobile phase A and 80% acetonitrile as mobile phase B Using an initial B concentration of 5%, linearly 30% in the first 15 minutes, then linearly 100% in 5 minutes, then mobile phase B at 100% and maintained for 5 minutes, then migrated The next sample was injected after 35 minutes with 5% phase B. DI0NEX Ultimate 3000 was used for feeding, and analysis was performed at a flow rate of 500 nL / min. The sample solution was injected 5 μm by CTC autosampler PAL, and the sample was once injected into the sampler loop of the injector and then fed into the analytical ram. Using ABI's Qstar XL equipped with an LC-MS interface made by Nihon Technos, 2.4 kV was printed as the ESI voltage. The measurement was performed in Information dependent acquisition mode, after a 1-second survey scan, followed by a maximum of 3 MSMS scans (0.6 seconds each). The switch from the MSMS mode power to the survey scan was 1 spectrum.
得られたデータについては、 Mascot (Matrixscience 社）及ぴ大腸菌（W3110)デ ータベース用いてタンパク質の自動同定を行った。 目的ピークの定量は MassNavigator (三井情報開発） を用いて行った。 R. Monicaらの報告（R. Monica et al, Nucleic Acid Research 34 (2006) 1)を参考にして、 TMHMM アルゴリズム で膜貫通領域を持つものを膜タンパク質とした。ぺプチドの GRAVY (grand average of hydropathy) スコアは Kyteらの方法 （J. Kyte, RF. Doolittle, J Mol. Biol 152 (1982) 105) に従って計算し、 スコアが正のものを疎水性ペプチド、 負のも のを親水性べプチドとした。 The resulting data were subjected to automatic identification of Mascot (Matrixscience Co.)及Pi coli (W 3 110) database used proteins. The quantification of the target peak is This was done using MassNavigator (Mitsui Information Development). With reference to a report by R. Monica et al. (R. Monica et al, Nucleic Acid Research 34 (2006) 1), a protein having a transmembrane region by the TMHMM algorithm was defined as a membrane protein. The peptide GRAVY (grand average of hydropathy) score was calculated according to the method of Kyte et al. (J. Kyte, RF. Doolittle, J Mol. Biol 152 (1982) 105). These were hydrophilic peptides.
結果を図 1に示す。 SDCに可溶化させた大腸菌の膜タンパク質画分について相 間移動法と酸沈殿法で大腸菌膜画分タンパク質の同定結果を比較したところ、 相 間移動法を用いることで酸沈殿法に比べて、 タンパク質及ぴぺプチドの同定数は 約 1. 4倍に増加した。 また、 同定できたべプチドの疎水性度を GRAVYスコアに従 い計算し、 疎水性が髙いとされる GRAVYスコアが 0より大きいぺプチドの数は約 1. 6倍に増加していた。 さらに、 膜タンパク質の同定数は約 2倍に増加した。 既 存の方法である 8M尿素を用いた方法に比べても、相間移動法を組み合わせた SDC を用いた方法では膜タンパク質の同定数が約 3倍増加し（SDC: 14、 尿素： 4)、 全体 では同定数が 5. 08倍増加した（SDC : 108、 尿素： 37)。 また、 同定できたペプチド の数を比べると、 SDCの方が尿素を用いるよりも、 約 5. 3倍増加した。  The results are shown in Figure 1. When comparing the identification results of the E. coli membrane fraction protein by the phase transfer method and the acid precipitation method for the membrane protein fraction of E. coli solubilized in SDC, compared to the acid precipitation method by using the phase transfer method, The number of identified proteins and peptides has increased by a factor of about 1.4. In addition, the hydrophobicity of the identified peptides was calculated according to the GRAVY score, and the number of peptides with a GRAVY score greater than 0, which is considered to be hydrophobic, increased approximately 1.6 times. In addition, the number of membrane proteins identified increased approximately 2-fold. Compared with the existing method using 8M urea, the method using SDC combined with the phase transfer method increases the number of identified membrane proteins by about 3 times (SDC: 14, urea: 4). Overall, the number of identifications increased 5.08 times (SDC: 108, urea: 37). When comparing the number of peptides that could be identified, SDC increased approximately 5.3 times compared to urea.
上記の通り、 本発明の相間移動法は、 従来の方法を用いて調製したタンパク質 試料に比較して、 同定されるタンパク質数及びぺプチド数の面で大きく向上する ことがわかった。  As described above, it was found that the phase transfer method of the present invention is greatly improved in terms of the number of proteins to be identified and the number of peptides as compared to the protein sample prepared by using the conventional method.
実施例 2：界面活性剤及び有機溶媒の組合せにおける液液分離評価 Example 2: Evaluation of liquid-liquid separation in combination of surfactant and organic solvent
本実施例では、 本発明の方法に種々の界面活性剤及び有機溶媒の組合せを使用 した際の液液分離を評価した。 なお、 本実施例では、 実施例 1で調製したタンパ ク質膜画分を不溶性タンパク質試料として使用し、 下記に記載する点を除いて実 施例 1の相間移動法と同様にして実施した。  In this example, liquid-liquid separation when a combination of various surfactants and organic solvents was used in the method of the present invention was evaluated. In this example, the protein membrane fraction prepared in Example 1 was used as an insoluble protein sample, and the same procedure as in Example 1 was performed except for the points described below.
本実施例で使用した界面活性剤と、 相間移動に使用した有機溶媒の組合せを下 記に示す。なおいずれの試料溶液も 50raM炭酸水素ァンモニゥムを添加して調製し た：  The combination of the surfactant used in this example and the organic solvent used for the phase transfer is shown below. All sample solutions were prepared by adding 50raM hydrogen carbonate:
界面活性剤 有機溶媒 組合せ 1. 10%ドデシル硫酸ナトリウム（SDS) ォクタノール（OctOH) 組合せ 2. 10ο /。コール酸ナトリウム ォクタノール 組合せ 3. 10%グリココール酸 才クタノーノレ 組合せ 4. 1%及び 10%ラウリルサルコシン酸ナトリウム ォクタノール 組合せ 5. 10%デォキシコール酸ナトリウム ォクタノール 組合せ 6. 10%デォキシコール酸ナトリウム へキサノール 組合せ 7. 1%及ぴ 10%デォキシコール酸ナトリウム 酢酸ェチル (EtAc) 組合せ 8. 1%及び 10%デォキシコール酸ナトリウム ジェチノレエーテノレ 組合せ 9. 1%及び 10%デォキシコール酸ナトリウム クロロホ/レム 組合せ 10. l%RapiGest™ SF ォクタノール 組合せ 11. 1 %及ぴ 10%グリコケノデォキシコール酸ナトリウム ォクタノール 組合せ 12. 10%ケノデォキシコール酸ナトリウム ォクタノール 組合せ 13. 10 %ウルソデォキシコール酸ナトリウム ォクタノール Surfactant Organic solvent Combination 1. 10% sodium dodecyl sulfate (SDS) Octanol (OctOH) Combination 2. 10 ο /. Sodium cholate octanol combination 3. 10% glycocholic acid pentanol combination 4. 1% and 10% sodium lauryl sarcosinate octanol combination 5. 10% sodium deoxycholate octanol combination 6. 10% sodium deoxycholate hexanol combination 7.1 % And 10% Sodium Deoxycholate Ethyl Acetate (EtAc) Combination 8.1% and 10% Sodium Deoxycholate Jetinoreethenole Combination 9. 1% and 10% Sodium Deoxycholate Chlorophore / REM Combination 10. l% RapiGest ™ SF Octanol Combination 11.1% and 10% Sodium glycochenodeoxycholate Octanol Combination 12. 10% Sodium kenodeoxycholate Octanol Combination 13. 10% Sodium urodeoxycholate Octanol
使用した上記界面活性剤及び有機溶媒の製造元を下記の表 1に示す。  The manufacturers of the surfactants and organic solvents used are shown in Table 1 below.
表 1  table 1
Figure imgf000019_0001
液液分離の評価は以下のように行った。 すなわち、試料中の界面活性剤を、 100 μ ΐの試料溶液に対して、 Ι μ ίのトリフルォロ酢酸 （TFA) (SDSの場合は 50 し の 1M KC1、 15mM Tris) を加えて脂溶化させた。 脂溶化界面活性剤は小型遠心機 で 5分間遠心分離して沈殿させ、 次いで、 上記組合せに対応した有機溶媒を添加 した。 その後、 ボルテックスをして、 小型遠心機で 5分間遠心分離し、 脂溶化界 面活性剤の溶解を評価した。
Figure imgf000019_0001
Evaluation of liquid-liquid separation was performed as follows. That is, the surface active agent in the sample, relative to 100 mu sample solution I, (in the case of SDS 1M KC1, 15 mM Tris 5 0 Mr) Ι μ ί of Torifuruoro acid (TFA) is fat soluble by adding It was. Fat-solubilized surfactant is a small centrifuge The mixture was centrifuged for 5 minutes to precipitate, and then an organic solvent corresponding to the above combination was added. Then, vortexed and centrifuged for 5 minutes in a small centrifuge to evaluate the dissolution of the fat-solubilized surfactant.
上記組合せ 1〜13における液液分離の結果をそれぞれ図 2〜14に示す。  The results of liquid-liquid separation in the above combinations 1 to 13 are shown in FIGS.
なお、 上記実験に関する追加情報は以下の通りである ：  Additional information regarding the above experiment is as follows:
組合せ 1において、 SDSの塩は 50uLの 1M KC1, 15m Trisで交換した。 In combination 1, the SDS salt was exchanged with 50 uL of 1M KC1, 15m Tris.
組合せ 4において、 10% ラゥリルサルコシン酸ナトリゥムは 10倍量の OctOHを加 えても除去できなかった。 In combination 4, 10% sodium lauryl sarcosinate could not be removed by adding 10 times the amount of OctOH.
組合せ 7において、 10% デォキシコール酸ナトリゥムは 10倍量の EtAcを加えて も除去できなかった。 In combination 7, 10% sodium deoxycholate could not be removed by adding 10 times the amount of EtAc.
組合せ 8において、 10% デォキシコール酸ナトリウムは 10倍量のジェチルエーテ ルを加えても除去できなかった。 In combination 8, 10% sodium deoxycholate could not be removed by adding 10 times the amount of jetyl ether.
組合せ 9において、 10% デォキシコール酸ナトリウムは 10倍量のクロ口ホルムを 加えても除去できなかった。 In combination 9, 10% sodium deoxycholate could not be removed by adding 10 times the amount of black mouthform.
組合せ 10において、 RapiGest™ SFは 0. 5% TFAとなるように添加し、 37°Cで 30 分間、 インキュベートして分解した。 In combination 10, RapiGest ™ SF was added to 0.5% TFA and incubated for 30 minutes at 37 ° C to degrade.
組合せ 11において、 ダリコケノデォキシコール酸ナトリゥムは 10%の濃度では完 全に溶けなかった。 In combination 11, sodium daricochenodeoxycholate was not completely dissolved at a concentration of 10%.
図からわかるとおり、 界面活性剤として 1%濃度のデォキシコール酸ナトリウ ムを使用した場合には、 有機溶媒の種類によらず、 消化反応溶液に対して 0. 5〜8 倍用量の有機溶媒に、 脂溶化デォキシコール酸ナトリゥムを完全に溶解すること ができた （図 5、 8〜： 12)。 また 10%濃度のデォキシコール酸ナトリウムを使用し た場合には、 消化反応溶液に対して 0. 5倍用量のオタタノール又は等量のへキサ ノールには溶解するが （図 6及ぴ 7)、 有機溶媒として酢酸ェチル、 ジェチルエー テル及びクロロホルムを使用する場合には、消化反応溶液に対して 10倍用量で用 いても完全には溶解しなかった （図 8〜： 10)。 これらの結果は、 脂溶化デォキシコ 一ル酸ナトリゥムは特にォクタノール又はクロロホルムに溶解し易く、 デォキシ コール酸ナトリゥムとォクタノール又はクロ口ホルムとの組合せが、 消化タンパ ク質の回収効率を向上し、 結果、 タンパク質の同定効率を向上させるために有用 であることを示唆する。 As can be seen from the figure, when 1% sodium dioxycholate was used as the surfactant, the organic solvent was used at 0.5 to 8 times the dose of the digestion reaction solution, regardless of the type of organic solvent. The fat-solubilized sodium doxycholate was completely dissolved (Fig. 5, 8 ~: 12). When 10% sodium deoxycholate is used, it dissolves in 0.5-fold dose of otanol or an equivalent amount of hexanol compared to the digestion reaction solution (Figures 6 and 7). When ethyl acetate, jetyl ether and chloroform were used as solvents, they were not completely dissolved even when used at a 10-fold dose to the digestion reaction solution (Figures 8 to 10). These results indicate that the fat-solubilized sodium doxycolate is particularly easily dissolved in octanol or chloroform, and the combination of sodium deoxycholate and octanol or black mouth form improves the recovery efficiency of the digested protein. Useful for improving protein identification efficiency It is suggested.
また有機溶媒としてのォクタノールの使用は、界面活性剤として 10%ラゥリル サルコシン酸ナトリゥム又は 10%ダリコケノデォキシコール酸ナトリゥムを使 用した場合を除き、 消化反応溶液に対して等量〜 3. 5 倍用量のォクタノールに脂 溶化界面活性剤を完全に溶解することができた （図 2〜5、 13〜14)。 これらの結 果は、 有機溶媒としてのォクタノールの使用は、 高濃度の脂溶化界面活性剤の溶 解に比較的適していることを示す。  The use of octanol as the organic solvent is equivalent to the digestion reaction solution except when 10% sodium lauryl sarcosinate or 10% sodium daricochenodoxycholate is used as a surfactant. It was possible to completely dissolve the fat-solubilized surfactant in 5 times the dose of octanol (Figures 2-5, 13-14). These results indicate that the use of octanol as the organic solvent is relatively suitable for dissolving high concentrations of fat-solubilized surfactants.
以上の結果から、 使用する界面活性剤の種類及ぴ量に応じて、 本発明の液液分 離に好適な有機溶媒との組合せが存在することが示唆された。 当業者は、 本発明 の開示に基づき、 適当な界面活性剤及びその濃度、 並びに有機溶媒の組合せを選 択することができる。 実施例 3：本発明の相間移動法において、 デォキシコール酸ナトリゥム、 ラゥ リルサルコシン酸ナトリゥムおよびその混合物を用いた溶液消化法におけるタン パク質同定結果の比較  From the above results, it was suggested that a combination with an organic solvent suitable for liquid-liquid separation of the present invention exists depending on the type and amount of the surfactant to be used. One of ordinary skill in the art can select a suitable surfactant and its concentration, and a combination of organic solvents, based on the present disclosure. Example 3: Comparison of protein identification results in solution digestion using sodium deoxycholate, sodium lauryl sarcosinate and mixtures thereof in the phase transfer method of the present invention
試料調製 Sample preparation
膜画分は、 実施例 1と同様にして、 大腸菌 K - 12株 BW25113から調製した。 膜タンパク質の可溶化剤には 120 raM (5%相当） デォキシコール酸ナトリウム The membrane fraction was prepared from E. coli K-12 strain BW25113 in the same manner as in Example 1. 120 raM (equivalent to 5%) sodium deoxycholate for membrane protein solubilizer
(SDC,和光純薬 Cat. No. 190-08313)、 24 mM N-ラゥリルサルコシン酸ナトリウム(SDC, Wako Pure Chemicals Cat.No. 190-08313), 24 mM sodium N-lauryl sarcosinate
(SLS, 和光純薬 Cat. No. 192- 10382)及ぴ 12 mM SDC- 12 mM SLS混合物を用いた。 採取した BW25113の膜画分に対して 200 i Lの可溶化剤を加えて懸濁した後、 95°C で 5分間加熱した。 膜タンパク質懸濁液は超音波発生器（T0CH0、 UC- 1331N)で 10 分間処理した。 10 Lの試料を用い、 1 Lの 10 mMのジチォスレイトール (和 光純薬 Cat. No. 040-29223： DTT) を加え、 室温で 30分インキュベーションして タンパク質中のシスティン残基を還元した。 その後、 55 mM のョードアセトアミ ド (和光純薬 Cat. No. 091-02153) を 1 し加え、 30分ィンキュベーションして システィン残基をアルキル化した。 120 mM SD 24 mM SLSを含む試料溶液は 50mM 炭酸水素アンモニゥム緩衝液で 10倍に、 12 mM SDC- 12 mM SLSを含む試料溶液は(SLS, Wako Pure Chemicals Cat. No. 192-10382) and 12 mM SDC-12 mM SLS mixture were used. To the collected membrane fraction of BW25113, 200 iL of solubilizer was added and suspended, and then heated at 95 ° C for 5 minutes. The membrane protein suspension was treated with an ultrasonic generator (T0CH0, UC-1331N) for 10 minutes. Using 10 L of sample, 1 L of 10 mM dithiothreitol (Wako Pure Chemicals Cat. No. 040-29223: DTT) was added and incubated at room temperature for 30 minutes to reduce cysteine residues in the protein. . Subsequently, 55 mM of odoacetamide (Wako Pure Chemicals Cat. No. 091-02153) was added to the mixture, and incubated for 30 minutes to alkylate cysteine residues. Sample solution containing 120 mM SD 24 mM SLS is 10 times with 50 mM ammonium bicarbonate buffer, and sample solution containing 12 mM SDC-12 mM SLS is
5倍に希釈した後、 0. 1 mg/mLのトリプシン (プロメガ社製、 Cat. No. V511C) を 2 L加え、 室温で 24時間インキュベーションし、 タンパク質を消化した。 SDC、 SLS は相間移動法で除去した。 相間移動法は試料と等量の酢酸ェチル（和光純薬 Cat. No. 055- 05991)を加え、 TFAを終濃度で 1%になるように添加した。 2液を 懸濁させた後、 13， 000回転で 2分間遠心分離して、 2相に分配した。 SDCを含む 酢酸ェチル相（上相）をピぺッティングで除去した。水相（下相）は減圧乾燥機（T0MY CC - 105)で 45分間処理した後、 以後の操作に用いた。 After 5-fold dilution, 0.1 mg / mL trypsin (Promega, Cat.No. V511C) was added. 2 L was added and incubated at room temperature for 24 hours to digest the protein. SDC and SLS were removed by the phase transfer method. In the phase transfer method, an equivalent amount of ethyl acetate (Wako Pure Chemicals Cat. No. 055- 05991) was added to the sample, and TFA was added to a final concentration of 1%. After the two liquids were suspended, they were centrifuged at 13,000 rpm for 2 minutes and distributed into two phases. The ethyl acetate phase (upper phase) containing SDC was removed by pipetting. The aqueous phase (lower phase) was treated with a vacuum dryer (T0MY CC-105) for 45 minutes and then used for the subsequent operations.
LC-MS測定 LC-MS measurement
実施例 1と同様にして、 LC (C18 column) /MS (ABI/QSTAR XL) システムを用 いての測定を η=3で行った。また、データ解析についても実施例 1と同様に行い、 η=3の結果を結合して解析した。  In the same manner as in Example 1, measurement using an LC (C18 column) / MS (ABI / QSTAR XL) system was performed at η = 3. Data analysis was performed in the same manner as in Example 1, and the results of η = 3 were combined and analyzed.
結果を図 15に示す。 SDCと SLSを混合することにより、 より多くのタンパク質 およぴ膜タンパク質を同定できることがわかった。 膜タンパク質における膜貫通 領域数についても比較を行ったところ、 こちらについても SDC— SLS混合系で、 よ り多くの膜貫通領域が同定されることが認められた（図 16)。 産業上の利用可能性  The results are shown in FIG. It was found that more proteins and membrane proteins could be identified by mixing SDC and SLS. A comparison of the number of transmembrane regions in membrane proteins also showed that more transmembrane regions were identified in the SDC-SLS mixed system (Fig. 16). Industrial applicability
本発明の方法によれば、 分析装置への影響が少ないタンパク質試料を調製する ことができる。 また本発明の方法により、 分析対象タンパク質をプロテアーゼ処 理した後のタンパク質消化画分の回収効率を格段に向上させることができる。 本明細書で引用した全ての刊行物、 特許および特許出願をそのまま参考として 本明細書にとり入れるものとする。  According to the method of the present invention, it is possible to prepare a protein sample with little influence on the analyzer. In addition, according to the method of the present invention, the recovery efficiency of the protein digested fraction after subjecting the protein to be analyzed to protease treatment can be significantly improved. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

PGT/JP 2008/068124WO 2009/044903 PCT/JP2008/068124 請求の範囲 PGT / JP 2008 / 068124WO 2009/044903 PCT / JP2008 / 068124 Claims
1 . (a) (i)分析対象タンパク質； （i i) l種又は 2種以上の脂溶性に改変可能 な界面活性剤；及び（i i i)プロテアーゼを含む反応溶液中で、分析対象タンパク質 を消化するステップと、 1. (a) (i) the protein to be analyzed; (ii) one or more lipophilic surfactants that can be modified; and (iii) digest the protein to be analyzed in a reaction solution containing a protease. Steps,
(b)該反応溶液に前記界面活性剤を脂溶化する試薬を添加するステップとを含 み、  (b) adding a reagent for fat-solubilizing the surfactant to the reaction solution,
脂溶化された前記界面活性剤を有機溶媒中に分離することを特徴とする、 タンパ ク質試料の調製方法。 A method for preparing a protein sample, comprising separating the fat-solubilized surfactant in an organic solvent.
2 . 脂溶性に改変可能な界面活性剤がィオン性界面活性剤であることを特徴 とする請求項 1記載の方法。  2. The method according to claim 1, wherein the surfactant capable of being modified to be lipophilic is an ionic surfactant.
3 . ィオン性界面活性剤はドデシル硫酸ナトリウム （SDS) であることを特徴 とする請求項 2記載の方法。  3. The method according to claim 2, wherein the ionic surfactant is sodium dodecyl sulfate (SDS).
4 . 前記界面活性剤を脂溶化する試薬はカリゥム塩であることを特徴とする 請求項 3記載の方法。  4. The method according to claim 3, wherein the reagent for fat-solubilizing the surfactant is a potassium salt.
5 . ィォン性界面活性剤は力ルボン酸型界面活性剤であることを特徴とする 請求項 2記載の方法。  5. The method according to claim 2, wherein the ionic surfactant is a strong rubonic acid type surfactant.
6 . 力ルポン酸型界面活性剤はコ一ル酸ナトリウム、 グリココール酸、 ラゥ リルサルコシン酸ナトリウム、 デォキシコール酸ナトリウム、 グリコケノデォキ シコール酸ナトリゥム、 ケノデォキシコール酸ナトリゥム又はウルソデォキシコ 一ル酸ナトリウムであることを特徴とする請求項 5記載の方法。  6. Strong ruponic acid type surfactants are sodium cholate, glycocholic acid, sodium lauryl sarcosinate, sodium deoxycholate, sodium glycochenodeoxycholate, sodium kenodeoxycholate or sodium ursodeoxycolate 6. The method of claim 5, wherein:
7 . 脂溶性に改変可能な界面活性剤が酸不安定界面活性剤であることを特徴 とする請求項 1記載の方法。  7. The method according to claim 1, wherein the surfactant capable of being modified to be lipophilic is an acid labile surfactant.
8 . 酸不安定界面活性剤は、 3-[ (2 -メチル- 2 -ゥンデシル- 1, 3-ジォキソラン -4 -ィル）メ トキシル] -1-プロパンスルホン酸ナトリ ゥム又は 3- [3- (1， 1-アルキ ルォキシェチル）ピリジン - 1 -ィル]プロパン- 1 -スルホン酸であることを特徴とす る請求項 7記載の方法。  8. Acid labile surfactants include 3-[(2-methyl-2-undecyl-1,3-dioxol-4-yl) methoxyl] -1-propanesulfonic acid sodium or 3- [3 8. A process according to claim 7, characterized in that it is-(1,1-alkyloxychetyl) pyridine-1-yl] propane-1-sulfonic acid.
9 . 前記界面活性剤を脂溶化する試薬は酸であることを特徴とする請求項 5 又は 7記載の方法。 PGT/JP 2008/0681249. The method according to claim 5 or 7, wherein the reagent for fat-solubilizing the surfactant is an acid. PGT / JP 2008/068124
WO 2009/044903 PCT/JP2008/068124 WO 2009/044903 PCT / JP2008 / 068124
1 0 . 前記酸はトリフルォロ酢酸であることを特徴とする請求項 9記載の方 法。 10. The method of claim 9, wherein the acid is trifluoroacetic acid.
1 1 . 2種以上の脂溶性に改変可能な界面活性剤は、 カルボン酸型界面活性 剤から選択されることを特徴とする請求項 1記載の方法。  1. The method according to claim 1, wherein the two or more types of surfactants that can be modified to be lipophilic are selected from carboxylic acid type surfactants.
1 2 . カルボン酸型界面活性剤は、 デォキシコール酸ナトリゥム及ぴラゥリ ルサルコシン酸ナトリゥムであることを特徴とする請求項 11記載の方法。  12. The method according to claim 11, wherein the carboxylic acid type surfactant is sodium deoxycholate and sodium lauryl sarcosinate.
1 3 . 前記有機溶媒は予め前記反応溶液中に混合されていることを特徴とす る請求項 1記載の方法。  13. The method according to claim 1, wherein the organic solvent is previously mixed in the reaction solution.
1 4 . 前記反応溶液はマイクロエマルジヨンの形態であることを特徴とする 請求項 13記載の方法。  14. The method of claim 13, wherein the reaction solution is in the form of a microemulsion.
1 5 . ステップ (b)後に前記有機溶媒を前記反応溶液に添加し、脂溶化された 前記界面活性剤を有機溶媒中に分離することを特徴とする請求項 1記載の方法。  15. The method according to claim 1, wherein the organic solvent is added to the reaction solution after step (b), and the fat-solubilized surfactant is separated into the organic solvent.
1 6 . 前記有機溶媒はォクタノール、 酢酸ェチル、 ジェチルエーテル、 クロ 口ホルム又はへキサノールであることを特徴とする請求項 1記載の方法。  16. The method according to claim 1, wherein the organic solvent is octanol, ethyl acetate, jetyl ether, chloroform, or hexanol.
1 7 . 分析対象タンパク質は電気泳動による分離後に切り出したゲルに含ま れる目的のタンパク質であることを特徴とする請求項 1記載の方法。  17. The method according to claim 1, wherein the protein to be analyzed is a protein of interest contained in a gel cut out after separation by electrophoresis.
1 8 . 請求項 1〜： 17のいずれか 1項記載の方法によりタンパク質試料を調製 し、 分析に供することを特徴とするタンパク質の分析方法。  18. A method for analyzing protein, comprising preparing a protein sample by the method according to any one of claims 1 to 17 and subjecting the sample to analysis.
1 9 . 前記分析は該サンプルを液体ク口マトグラフィ一質量分析（LC- MS) 又 はマトリックス支援レーザー脱離ィオン化質量分析 （MALDI- MS) であることを特 徴とする請求項 18記載のタンパク質の分析方法。  19. The analysis according to claim 18, wherein the analysis is liquid chromatography matrix mass spectrometry (LC-MS) or matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS). Protein analysis method.
PCT/JP2008/068124 2007-10-02 2008-09-30 Method of preparing protein sample WO2009044903A1 (en)

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JP2014039497A (en) * 2012-08-22 2014-03-06 Keio Gijuku Cell fractionation method using solution comprising ethylene glycol and surfactant
JP2015017813A (en) * 2013-07-09 2015-01-29 第一三共株式会社 Complete automation method for distribution coefficient measurement using automatic dispenser
WO2020214777A1 (en) * 2019-04-17 2020-10-22 Regeneron Pharmaceuticals, Inc. Identification of host cell proteins
US20210263040A1 (en) * 2013-10-24 2021-08-26 University Of Leeds Method and device for protein preparation
CN114137099A (en) * 2021-10-28 2022-03-04 中科新生命(浙江)生物科技有限公司 SF-based modified proteomics analysis method

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014039497A (en) * 2012-08-22 2014-03-06 Keio Gijuku Cell fractionation method using solution comprising ethylene glycol and surfactant
JP2015017813A (en) * 2013-07-09 2015-01-29 第一三共株式会社 Complete automation method for distribution coefficient measurement using automatic dispenser
US20210263040A1 (en) * 2013-10-24 2021-08-26 University Of Leeds Method and device for protein preparation
WO2020214777A1 (en) * 2019-04-17 2020-10-22 Regeneron Pharmaceuticals, Inc. Identification of host cell proteins
CN114137099A (en) * 2021-10-28 2022-03-04 中科新生命(浙江)生物科技有限公司 SF-based modified proteomics analysis method

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