JP2007309695A - Extraction method of lipid-modified peptide for mass analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for analyzing with a small amount of lipoprotein. <P>SOLUTION: A method for extracting lipid modified peptide from a sample, which contains the lipid-modified peptide and lipid-unmodified peptide, by extracting the sample with an organic solvent and a lipoprotein analysis method using the extraction method are disclosed, where the lipoprotein analysis method includes a process (a) for treating a lipoprotein-containing material with protease, a process (b) for extracting the sample treated with protease obtained in the process (a) with the organic solvent, and a process (c) for subjecting the extract obtained in the process (b) to mass analysis. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for extracting lipid-modified peptides and a method for analyzing lipoproteins.

  In order to promote the attachment of soluble proteins to biological membranes, post-translational modification of proteins by lipids is universally found in all organisms. Bacterial lipoproteins are proteins whose N-terminal cysteine residues are modified with lipids, and play essential functions such as maintaining the membrane structure at the membrane-water interface.

It has been known that lipid modification is indispensable for such functions, but recently, the structure of lipid modification dramatically changes the physiological activity of the protein, but it does not affect the membrane anchoring ability. It has become clear. One of the most well-studied examples of lipid modification is lipid modification of low molecular weight GTPases such as Ras or RhoB. The CAAX motif of the Rho / Ras GTPase family is known to be modified by either of two types of isoprenoid lipids, namely, geranylgeranyl group (C ₂₀ ) and farnesyl group (C ₁₅ ). These two types of modifications allow the association of GTPase to the biological membrane. However, geranylgeranylated RhoB and farnesylated RhoB show different physiological activities. Farnesylated RhoB is a growth promoting protein, whereas geranylgeranylated RhoB is an apoptosis-inducing protein. Ras is normally farnesylated and localizes to the plasma membrane. However, when mutated to the geranylgeranylated form, geranylgeranylated Ras will be localized in the intracellular membrane system.

  In this way, research on the relationship between lipid modification structure and physiological function has become very important in recent years. In eukaryotes, specific inhibitors and mutations of amino acids involved in lipid modification are used. Research has been done. However, lipid modification in prokaryotes such as bacteria has not been studied due to technical difficulties. This is because lipoproteins in prokaryotes are relatively small and difficult to purify. Therefore, a means capable of analyzing even a small amount of lipoprotein has been desired.

  There are mainly two methods for analyzing the structure of lipoprotein lipid modification. One is a chemical analysis method using TLC or gas chromatography, and the other is a physicochemical mass spectrometry method using LC-MS, FAB-MS, MALDI-TOF MS, or the like.

  Chemical analysis methods have been widely used in the past, but require a large amount of purified protein in millimolar amount. On the other hand, lipoproteins are not amenable to purification because of their amphiphilic nature and are not suitable for analysis of many lipoproteins including PsbQ and Psb27 derived from the cyanobacterium Synechocystis sp. PCC6803. Many lipoproteins including PsbQ and Psb27 are only known by gel separation and purification in micromolar amounts by SDS-PAGE or the like, and sufficient amounts cannot be recovered.

  On the other hand, mass spectrometry requires a smaller amount of sample than a general chemical analysis method, and an accurate molecular weight of protein can be obtained. However, in the analysis of lipoproteins using mass spectrometry, lipid-modified peptide fragments could not be detected because long alkyl chains derived from fatty acids inhibit protein ionization in mass spectrometry. In addition, when the lipoprotein separated by gel by SDS-PAGE or the like is subjected to mass spectrometry, there is a problem that hydrophobic lipoprotein or lipid-modified peptide cannot be recovered from the gel.

  The object of the present invention is to provide a means for analyzing lipoproteins in small amounts.

  As a result of intensive studies to solve the above problems, the present inventors have been able to separate lipid-modified peptides from a group of peptides obtained by hydrolyzing lipoproteins by organic solvent extraction. The present inventors have found that the lipoprotein separated by gel electrophoresis can be subjected to mass spectrometry by combining with an agent, and the present invention has been completed.

That is, the present invention includes the following inventions.
(1) A method for extracting a lipid-modified peptide from a sample by extracting the sample containing a lipid-modified peptide and a peptide not having a lipid modification with an organic solvent.
(2) The method according to (1), wherein the organic solvent is an organic solvent containing a low polarity organic solvent and a high polarity organic solvent.
(3) (a) Protease treatment of a sample containing lipoprotein
(b) A step of extracting the sample treated with the protease in the step (a) with an organic solvent
(c) A method for analyzing lipoprotein, comprising a step of subjecting the extract obtained in the step (b) to mass spectrometry.
(4) The method according to (3), wherein, in the step (a), a protease is treated with a supernatant obtained by adding a surfactant to a sample containing lipoprotein and centrifuging the sample.
(5) The method according to (3) or (4), wherein a lipid bound to a protein is analyzed.
(6) The method according to any one of (3) to (5), wherein the organic solvent is an organic solvent containing a low polarity organic solvent and a high polarity organic solvent.
(7) (a) A step of subjecting a sample containing lipoprotein to gel electrophoresis
(b) A step of subjecting the gel after electrophoresis to protease treatment
(c) A step of extracting a protease-treated sample with a solution containing a surfactant
(d) A step of further extracting the extract obtained in the step (c) with an organic solvent.
(e) A method for analyzing lipoprotein, comprising a step of subjecting the extract obtained in the step (d) to mass spectrometry.
(8) The method according to (7), wherein the surfactant is a nonionic surfactant.
(9) The method according to (7) or (8), wherein the organic solvent is an organic solvent containing a low polarity organic solvent and a high polarity organic solvent.
(10) The method according to any one of (7) to (9), wherein lipids bound to the protein are analyzed.

  According to the present invention, lipid-modified peptides can be extracted from a sample preferentially, and lipid-modified proteins including lipoproteins can be analyzed in a small amount.

  The present inventors have found that lipid-modified peptides are preferentially extracted into an organic solvent by extracting a sample containing both lipid-modified peptides and non-lipid-modified peptides with an organic solvent. It was. Therefore, in one embodiment, the present invention relates to a method for extracting a lipid-modified peptide from a sample by extracting the sample containing the lipid-modified peptide and the non-lipid-modified peptide with an organic solvent.

  As used herein, a lipid-modified peptide refers to a complex of a lipid and a peptide. Lipids include esters of fatty acids with various alcohols and their simple lipids (neutral lipids), such as fats and oils (triacylglycerol), waxes (fatty acid esters of higher alcohols), sterol esters, cholesterol esters, vitamins Fatty acid esters, etc .; in addition to fatty acids and alcohols, complex lipids having polar groups such as phosphoric acid, sugar, sulfuric acid, amines, such as phospholipids (glycerophospholipids, sphingophospholipids, etc.) and glycolipids (glyceroglycolipids, sphingosaccharides) Lipids, etc.); derived lipids that represent fat-soluble compounds produced by hydrolysis of simple lipids and complex lipids, such as fatty acids, higher alcohols, fat-soluble vitamins, steroids, hydrocarbons and the like.

  The present invention is particularly suitable for extracting peptides modified with lipids containing fatty acids. Fatty acids containing fatty acids include the above simple lipids, complex lipids and fatty acids, preferably glycerol fatty acid esters (monoacylglycerol, diacylglycerol, triacylglycerol) and lipids. Examples of the fatty acid include long-chain saturated fatty acids and long-chain unsaturated fatty acids having 8 or more carbon atoms, preferably 10 to 30 carbon atoms. Specific examples include decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, palmitoleic acid, methylenehexadecanoic acid, vaccenic acid, linoleic acid, linolenic acid, arachidonic acid and the like. It is done. According to the present invention, a lipid-modified peptide having a fatty acid at the N-terminus of the peptide can be suitably extracted.

  In the lipid-modified peptide that can be extracted by the present invention, the amino acid chain length of the peptide is not particularly limited, but is usually 1 to 50 amino acids, preferably 1 to 40 amino acids.

  The extraction method of the present invention is suitably used for extraction of lipid-modified peptides, that is, peptides to which lipids are bound, among a group of peptides obtained when lipoproteins are decomposed with a protease or the like.

  In the present specification, lipoprotein refers to a complex (lipid-modified protein) in which a protein is covalently bound to a lipid. Lipid-modified proteins include both natural proteins and synthetic modified proteins that are an artificial combination of lipids and proteins. Since the extraction method of the present invention is advantageously used in the analysis of lipid components in natural lipoproteins, it is particularly suitably used for extracting lipid-modified peptides from hydrolysates of natural lipoproteins. .

  The sample containing lipid-modified peptide and non-lipid-modified peptide is preferably a hydrolyzate of the lipoprotein, more preferably a protease-treated product of lipoprotein, and the lipoprotein is preferably a natural lipoprotein.

  As a protease-treated product of lipoprotein, a sample containing lipoprotein treated with protease can be used. Samples containing bacterial lipoproteins include bacterial cell membranes and outer membranes, and suspensions, crushed materials and extracts thereof, and purified lipoproteins obtained from these suspensions. Samples containing lipid-modified proteins other than bacterial lipoproteins may be animal-derived samples or plant-derived samples. For example, body fluids (eg, blood, plasma, serum, urine, sweat, tears, spinal fluid, ascites, lymph) ), Tissues, cells, cell cultures, suspensions, disruptions and extracts thereof, and purified products of lipid-modified proteins obtained therefrom.

Specific examples of lipid-modified proteins include bacterial lipoproteins (such as E. coli Lpp and Pal), pancreatic b-cell protein kinase C and Ca ²⁺ -ATPase, and other palmitoylated proteins, as well as eukaryotic cell growth control. Examples include proteins that are farnesylated or geranylgeranylated, such as Ras and Rho involved. Since the method of the present invention can effectively extract a small amount of lipid-modified peptide, it is also suitably used when extracting a lipid-modified peptide from a prokaryotic-derived lipoprotein hydrolyzate that is present only in a small amount.

  The protease used for the protease treatment is not particularly limited as long as it can hydrolyze the protein, and preferably a protease that specifically hydrolyzes the protein as usually used in the peptide fingerprint method. Specific examples include trypsin, lysyl endopeptidase, papain, pepsin and subtilisin, and trypsin is preferably used.

  Lipid-modified peptides are water-soluble and have not previously been thought to migrate to the phase of organic solvents, but the present inventors have found that lipid-modified peptides can be extracted preferentially with organic solvents. . As the organic solvent for extracting the lipid-modified peptide, one that is separated into two layers when added to an aqueous solution is used. It is preferable to use an organic solvent having polarity. Examples of the organic solvent having polarity include low polar organic solvents such as dichloromethane, chloroform, dichloroethane, trichloroethylene, and tetrachloroethylene, and high polar organic solvents such as lower alcohols having 1 to 6 carbon atoms (for example, methanol, ethanol, butanol and propanol), As well as mixtures thereof.

  Preferably, an organic solvent containing a low polarity organic solvent and a high polarity organic solvent is used. Particularly preferably, an organic solvent containing chloroform and methanol is used. When an organic solvent containing chloroform and methanol is used, the volume ratio of chloroform to methanol is usually 10: 1 to 5: 3, preferably 3: 1 to 2: 1. The organic solvent containing chloroform and methanol may contain other organic solvents such as diethyl ether and acetone.

According to the extraction method of the present invention, it has become possible to effectively extract lipid-modified peptides that have been difficult to extract by a simple method.
The present invention also relates to a method for analyzing lipoproteins using the method for extracting lipid-modified peptides described above.

In one embodiment, the present invention provides:
(a) subjecting a sample containing lipoproteins to protease treatment;
(b) A step of extracting the sample treated with the protease in the step (a) with an organic solvent
(c) The present invention relates to a method for analyzing lipoprotein, comprising a step of subjecting the extract obtained in the step (b) to mass spectrometry.

  The sample containing the lipoprotein in the step (a) is the same as described for the extraction of the lipid-modified peptide. Preferably, an extract of cell lysate is used. More preferably, a supernatant obtained by adding a surfactant to a sample containing lipoprotein as described above and centrifuging is used as the sample. Since the lipoprotein is water-soluble, the supernatant thus obtained contains the lipoprotein.

Examples of the surfactant added here include cationic surfactants, nonionic surfactants, and nonionic surfactants. Preferably, the surfactants are suitable for extraction of hydrophobic peptides without inhibiting peptide ionization. Use things. Nonionic surfactants are preferred. Examples of the nonionic surfactant include glycosides such as alkyl glycosides and alkylthioglycosides, RO (CH ₂ CH ₂ O) _n H (wherein R represents a lipophilic group, and n is 1 to 20). A compound represented by an integer) (for example, polyoxyethylene alkyl ether, polyoxyethylene alkylphenol), RCOO (CH ₂ CH ₂ O) _n H (wherein R represents a lipophilic group, n is 1 to A sorbitan fatty acid ester (for example, sorbitan monoisostearate and sorbitan sesquioleate), an organic acid glyceride (for example, succinic acid monoglyceride), a glycerin fatty acid ester (for example, pentaglycerin olein) Acid ester), propylene glycol fatty acid ester (for example, propylene glycol monostearate), sucrose fatty acid ester, curing Machine oil derivatives, glycerin alkyl ether.

  In the present invention, alkyl glycosides and alkylthioglycosides are preferably used. The alkyl glycoside refers to an acetal in which a hydrogen atom of a monosaccharide or oligosaccharide hemiacetal hydroxy group is substituted with an alkyl group having 8 to 30 carbon atoms. Alkylthioglycoside refers to an acetal in which a hemiacetal hydroxy group of a monosaccharide or oligosaccharide is substituted with an alkylthio group having 8 to 30 carbon atoms. Examples of the sugar moiety in the alkyl glycoside and alkyl thioglycoside include glucose, fructose, maltose, cellobiose, gentiobiose, lactose, galactose, trehalose, sucrose, and the like. Specific examples of alkyl glycosides and alkylthioglycosides include, for example, dodecylmaltopyranoside, dodecylthiomaltopyranoside, dodecylglucopyranoside, dodecylthioglucopyranoside, decylmaltopyranoside, decylthiomaltopyranoside, decylglucopyranoside, decyl Thioglucopyranoside, nonylmaltopyranoside, nonylthiomaltopyranoside, nonylglucopyranoside, nonylthioglucopyranoside, octylglucopyranoside, octylthioglucopyranoside, octylmaltopyranoside, octylthiomaltopyranoside, heptylglucopyranoside, heptylthioglucopyranoside, Examples include heptyl maltopyranoside and heptyl thiomaltopyranoside.

  In the protease treatment, a protease usually used in peptide fingerprinting, particularly trypsin, can be used in the same manner as described for extraction of lipid-modified peptides.

  Subsequently, in the step (b), the sample treated with the protease in the step (a) is extracted with an organic solvent. Since the protease-treated sample contains lipid-modified peptide and non-lipid-modified peptide, the lipid-modified peptide can be preferentially extracted by extraction with an organic solvent as described above. . The organic solvent is the same as described above.

  Subsequently, in the step (c), the extract obtained in the step (b) is subjected to mass spectrometry. By this step, the lipid-modified peptide contained in the extract obtained in the step (b) can be subjected to mass spectrometry. In the method for analyzing lipoproteins of the present invention, mass spectrometry may also be performed on peptides not modified with lipids extracted by conventional methods.

  In this specification, mass spectrometry refers to a technique of analyzing atomic / molecular ions based on the difference in mass using electrical interaction, and includes three steps of ion generation, separation, and detection. Such mass spectrometry is not particularly limited, and those known in the art can be used. The ionization methods that can be used for mass spectrometry include laser ionization, especially matrix-assisted laser desorption ionization (MALDI), electron impact ionization (EI), electrospray ionization (ESI), and photoionization. Examples include secondary ionization, fast atom bombardment ionization, field ionization ionization, surface ionization ionization, chemical ionization (CI), field ionization (FI), and ionization by spark discharge. A deionization (MALDI) method is preferred. In addition, separation modes include time-of-flight (TOF), single or multiple quadrupole, single or multiple magnetic sector, Fourier transform ion cyclotron resonance (FTICR), ion capture, radio frequency and ion capture. / Time-of-flight type, and the like, and those using the time-of-flight type are preferable. Mass spectrometry can be performed by combining the ionization method as described above with a detection mode such as separation mode, electrical recording and photographic recording. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is preferably used.

In another embodiment, the present invention provides:
(a) Step of subjecting a sample containing lipoprotein to gel electrophoresis
(b) A step of subjecting the gel after electrophoresis to protease treatment
(c) A step of extracting a protease-treated sample with a solution containing a surfactant
(d) A step of further extracting the extract obtained in the step (c) with an organic solvent.
(e) The present invention relates to a method for analyzing lipoprotein, comprising a step of subjecting the extract obtained in the step (d) to mass spectrometry.

  The sample containing the lipoprotein in the step (a) is the same as described for the extraction of the lipid-modified peptide. Preferably, an extract of cell lysate is used. More preferably, a supernatant obtained by adding a surfactant to a sample containing lipoprotein as described above and centrifuging is used as the sample.

  As gel electrophoresis, those commonly used for protein separation can be used. Preferably polyacrylamide gel electrophoresis is used. SDS-PAGE is widely used for proteome analysis, and many lipoproteins, including proteins from the cyanobacteria Synechocystis sp. PCC6803, PsbQ, Psb27, NrtA, Escherichia coli Escherichia coli, Pal, and Lpp Since it has been reported that separation and purification can be performed by -PAGE, SDS-PAGE is preferably used.

  In the step (b), the gel after the electrophoresis is treated with a protease. Specifically, a band that can contain the target lipoprotein is cut out from the gel after electrophoresis, and hydrolyzed with a protease, preferably trypsin.

  In step (c), the protease-treated sample is extracted with a solution containing a surfactant. The surfactant used here is the same as that used in the above embodiment.

  As the solution containing the surfactant, an aqueous solution having a surfactant concentration of 0.3 (w / v)% to 1.0 (w / v)% is preferably used. By using in this concentration range, the influence on mass spectrometry in the subsequent step (e) can be reduced.

  In the step (d), the obtained extract is further extracted with an organic solvent. The organic solvent used here is also the same as the organic solvent used for extraction of the lipid-modified peptide described above.

  Conventionally, it has been difficult to recover lipoproteins and lipid-modified peptides from gels after electrophoresis, but by combining extraction with surfactants and extraction with organic solvents by the method of the present invention, It was possible to perform mass spectrometry on the attached lipoprotein.

  Subsequently, in the step (e), the extract obtained in the step (d) is subjected to mass spectrometry. Mass spectrometry is the same as in the above embodiment. Furthermore, mass spectrometry may also be performed on peptides that are not lipid-modified extracted by conventional methods.

  By mass-analyzing a peptide group obtained by treating lipoproteins with a prosthesis by the method of the present invention, lipoproteins can be identified by mass fingerprinting. That is, the lipoprotein can be identified by measuring the mass of ions derived from a large number of digested fragments (peptides) contained in the enzymatic degradation product of lipoprotein and comparing it with a known database.

  Examples of databases for identifying peptides include Mascot, MS-Tag, Peptide Search, PepFrag, and SEQUEST (experimental medicine separate volume, post-genome era experimental course 2, proteome analysis method, Yodosha (2000) )).

  In the method for analyzing lipoproteins of the present invention, the mass of lipid-modified peptides contained in the lipoprotein protease-treated product can be analyzed with high resolution, so that lipid components in lipoproteins can be analyzed and identified. . Therefore, it is a useful tool in research on the relationship between lipid modification and function of proteins.

  Conventionally, a large amount of sample has been required for the analysis of lipoprotein, but by analyzing the lipoprotein using the analysis method of the present invention, it is possible to analyze even when a small amount of lipoprotein is used. become.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the scope of the present invention is not limited to an Example.

The source of chemical substances used in this example is as follows:
Ni-NTA agarose, Qiagen (Valencia, CA) from; C ₄ and C ₁₆ ZIP TIP (TM), Millipore (Bedford, MA) from; is reductively alkylated sequencing grade trypsin, Promega (From Madison, WI); trifluoroacetic acid (TFA) and sinapinic acid from Nacalai Tesque (Kyoto, Japan); α-cyano-4-hydroxycinnamic acid (CHCA) and acetonitrile from Sigma Aldrich (St Louis, St. MO); all other chemical reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan) unless otherwise stated.

Example 1 Purification of PsbQ-His expressed in E. coli PsbQ of cyanobacteria Synechocystis sp. PCC6803 (Thornton LE, Ohkawa H, Roose JL, Kashino Y, Keren N, Pakrasi HB (2004) Homologs of plant PsbP and PsbQ proteins are necessary DNA region encoding for regulation of photosystem II activity in the cyanobacterium Synechocystis 6803. Plant Cell 16: 2164-2175) Amplified by PCR using SEQ ID NO: 2). Nucleotide sequences containing an NcoI site and a HindIII site, 5′-CATGCCATGG-3 ′ (SEQ ID NO: 3) and 5′-CCCAAGCTT-3 ′ (SEQ ID NO: 4), respectively, were added to the 5 ′ end of each primer. The PCR product was digested with NcoI and HindIII and ligated to the NcoI-HindIII site of the expression vector pBAD / Myc-HisB (pBAD) (Invitrogen, Carlsbad, Calif.) To obtain the desired in-frame product. This plasmid was called pBAD-PsbQ-His and was used to transform E. coli JM109.

  Transformed cells with pBAD-PsbQ-His were grown at 37 ° C. in LB medium. When the turbidity at 600 nm reached 0.5, 0.2% arabinose was added to the growth medium to induce the expression of PsbQ-His and further incubated for 3 hours. Cells were harvested after incubation. The collected cells were suspended in 20 mM Tris-HCl (pH 7.5) and disrupted by sonication. Cell debris was centrifuged at 100,000 g for 10 minutes to obtain a total membrane fraction containing PsbQ-His. Next, the membrane fraction was solubilized using 1% n-dodecyl β-D-maltopyranoside (DM) and centrifuged at 100,000 g for 15 minutes to obtain a solubilized protein as a supernatant.

  The supernatant was then added to a 3 ml Ni-NTA agarose column pre-equilibrated with 2 volumes of 20 mM Tris-HCl (pH 7.5) containing 1% DM and incubated at 4 ° C. for 1 hour. After washing well with 20 mM Tris-HCl (pH 7.5), 1% DM and 20 mM imidazole buffer, the protein was eluted from the column with 20 mM Tris-HCl (pH 7.5), 1% DM and 200 mM imidazole. . The eluted protein was precipitated with 10% trichloroacetic acid and washed with acetone and diethyl ether. About 0.5 mg of PsbQ-His was obtained from 1 liter of culture solution.

Example 2 Analysis of PsbQ-His First, using a MALDI-TOF mass spectrometer (AXIMA-CFR; manufactured by Shimadzu Corporation), it was confirmed whether PsbQ-His obtained in Example 1 was modified with lipid. Measurements were made in positive linear mode.

  FIG. 2 shows the mass spectrum of PsbQ-His. From the calculated theoretical mass of PsbQ-His, a signal is observed at about 17,918 (m / z), which corresponds to PsbQ-His modified by lipids. This indicates that PsbQ-His expressed in E. coli was modified with lipids.

  Next, the fatty acid composition of the S-diacylglyceryl group bound to the SH group of the N-terminal cysteine residue of PsbQ-His was analyzed by gas chromatography. PsbQ-His precipitated and purified by TCA was washed 3 times with chloroform / methanol to remove the surfactant and membrane lipid. Next, the washed PsbQ-His was treated with HCl / methanol to produce a fatty acid methyl ester. Fatty acid methyl esters were extracted with n-hexane and analyzed by gas chromatography. The gas chromatogram shows the fatty acid composition of PsbQ-His (Figure 3). In the chromatogram, 16: 0, 16: 1, 17: 0 cyclo and 18: 1 (11) were detected. 16: 0 (57.4%) and 16: 1 (23.8%) were the main components of fatty acids. The fatty acid composition of diacylglycerol bound to PsbQ-His was calculated. 16: 1/16: 0, 17: 0 cyclo / 16: 0, and 18: 1/16: 0 were calculated to be most abundant.

Example 3 Preparation of PsbQ-His for MALDI-TOF MS analysis 5 μg of the purified PsbQ-His obtained in Example 1 was diluted with 1 μg in 20 μl of 40 mM ammonium bicarbonate containing 10% acetonitrile. Digested with trypsin.

Apply trypsinized product obtained separated by column (containing trypsin digestion fragments) to C ₄ and C ₁₆ ZipTip column (Millipore (Bedford, MA)), and washed several times with 0.1% TFA (trifluoroacetic acid) solution . Ziptip is known to be ideal for concentrating and purifying peptides or proteins prior to mass spectrometry, HPLC, capillary electrophoresis and other analytical techniques. Next, elution was carried out with a mixture of 0.1% TFA aqueous solution and 0.1% TFA acetonitrile solution in different ratios and other organic solvents such as DMF, and each eluate was eluted with 2 μl of matrix solution (0.1% TFA, 4-hydroxy- in 30% acetonitrile saturated with α-cyanocinnamic acid) and spotted on a MALDI-plate.

On the other hand, the trypsin-treated product (including the trypsin-digested fragment) obtained was extracted with 50 μl of chloroform / methanol (volume ratio 2: 1, the same applies hereinafter). After evaporating the chloroform / methanol, the extracted fragment is dissolved in 2 μl of matrix solution (0.1% TFA, 30% acetonitrile saturated with 4-hydroxy-α-cyanocinnamic acid) and spotted on a MALDI-plate did.

  The above two types of samples (column separation and organic solvent extraction) and samples that were neither subjected to column separation nor organic solvent extraction were subjected to mass spectrometry using a MALDI-TOF mass spectrometer (AXIMA-CFR; manufactured by Shimadzu Corporation). . To analyze trypsin digested fragments, measurements were made in positive reflection mode.

  First, FIG. 4A shows a mass spectrum of MALDI-TOF MS of trypsin digested PsbQ-His when neither column separation nor extraction with an organic solvent is performed. Many T ions were observed, but no signal of lipid-modified N-terminal fragment was observed. Lipid-modified fragments are known not to be ionized effectively because long alkyl chains in the lipid inhibit ionization, and the energy of ionization may have been used to ionize other fragments.

It is C ₁₈ or with C ₄ ZipTip column eluting with 100% acetonitrile 0.1% TFA, N-terminal fragment will not effectively eluted, from the N-terminal fragment which are lipid-modified by MALDI-TOF MS No signal was detected. Elution with DMF (dimethylformamide) and other organic solvents also had no effect. These results, N-terminal fragment that is lipid modification is not able to recover from the C ₄ or C ₁₈ column.

  On the other hand, when a fragment extracted with an organic solvent (chloroform / methanol) was applied to MALDI-TOF MS, it was derived from a lipid-modified N-terminal fragment that did not exist at all in the previous spectrum, as shown in FIG. 4B. A signal was observed. This indicates that the lipid modified fragment was effectively extracted by chloroform / methanol extraction.

  This type of extraction has never been performed because the lipid-modified fragment was water soluble and was not thought to migrate into the organic solvent phase. However, lipid modified fragments have been shown to have a higher affinity for organic solvents than water.

FIG. 5 shows the mass spectrum of the lipid-modified N-terminal fragment. Signals were detected in 6 regions denoted I-III and I ^* -III ^* . According to the calculated mass signal of the region, region I, region II and region III are 2 palmitic acids and 1 palmitoleic acid (16: 0/16: 1/16: 0) and 2 respectively. Of palmitic acid and 1 cis-9,10-methylenehexadecanoic acid (16: 0/17: 0 cyclo / 16: 0), and 2 palmitic acid and 1 vaccenic acid [16: 0/18: Corresponds to ions derived from N-terminal fragments modified with lipids including 1 (11) / 16: 0]. I ^* , II ^* and III ^* indicate ions derived from the sodium adduct of the respective N-terminal fragment.

  From the calculated theoretical molecular weight, a signal of 2,551 (m / z) corresponds to a diacylglycerol containing three fatty acids (16: 0/16: 1/16: 0) and a fragment modified with an acyl group To do. Signals were also detected at 2,565 (m / z) (2,551 + 14) and 2,579 (m / z) (2,551 +28), indicating that other fatty acids are bound to the fragment. From the calculated theoretical mass, a signal of 2,566 (m / z) corresponds to an N-terminal fragment containing 3 fatty acids (16: 0/17: 0 cyclo / 16: 0), 2,579 (m / z ) Signal corresponds to an N-terminal fragment containing fatty acids (16: 0/18: 1 (11) / 16: 0). Another signal (+22 m / z) corresponding to the sodium adduct of each signal was also detected. This result is consistent with the result obtained by gas chromatography analysis in Example 2. These results indicate that the method of the present invention provides high resolution for determining fatty acids that bind to lipid-modified N-terminal fragments.

Example 4 MS / MS analysis In order to confirm the structure of lipid modification, MS / MS analysis of lipid-modified N-terminal fragment was performed. For MS / MS analysis, a MALDI quadrupole ion trap time-of-flight mass spectrometer (AXIMA-QIT; manufactured by Shimadzu Corporation) was used.

  The mass / mass spectrum of the signal detected at 2,551 (m / z) in the mass spectrum of MALDI-TOF MS of Example 3 is shown in FIG. Many y ions from the N-terminal fragment of PsbQ-His were observed, indicating that the 2,551 (m / z) signal is that of the N-terminal fragment. However, b ions were not observed. This indicates that the long alkyl chain of fatty acid binding to the N-terminal cysteine residue inhibited ionization. The MS / MS spectrum of the 2,565 (m / z), 2,579 (m / z) or other sodium type signal showed a spectrum similar to that of the 2,551 (m / z) signal. Therefore, these signals were confirmed to be derived from the lipid-modified N-terminal fragment of PsbQ-His, and the different molecular weights reflect the composition of fatty acids that bind to PsbQ-His.

Example 5 Preparation of PsbQ-His from a polyacrylamide gel Purified PsbQ-His was prepared using a 12% polyacrylamide gel (thickness 1.5 mm) according to the method of Laemmli UK (1970), Nature 227: 680-685. SDS-PAGE was performed. Since the Tris buffer system had to be used in the destaining method (Lee C, Levin A, Branton D (1987) Biochem. 166: 308-312), Tris-Glycine buffer was used. Electrophoresis was performed at 100 V, and was completed when the tip of the dye reached the bottom of the gel. Following electrophoresis, proteins were visualized by copper staining (Lee et al., 1987, supra). A band corresponding to PsbQ-His was scraped from the gel using a clean microspatula. The gel slice was then transferred to a 1.5 ml tube for destaining. Destaining was performed using Tris-Glycine destaining solution (Bio-Rad, Hercules, CA). Destained gel slices were dehydrated by incubating twice in a solution of 50% methanol and 50 mM ammonium bicarbonate for 15 minutes at 40 ° C. After incubation, the gel slices were crushed and dried in a vacuum centrifugal concentrator at room temperature for 10-15 minutes to remove methanol. During vacuum centrifugal concentration, trypsin was dissolved in 50 mM acetic acid at a concentration of 1 mg / ml, diluted to 20 μg / ml with digestion solution (40 mM ammonium bicarbonate / 10% acetonitrile), and the trypsin solution was diluted. Had made. Dry gel slices were placed in a minimal amount of trypsin solution (10-20 μl) and preincubated for 1 hour under ice cooling to allow trypsin to soak into the gel. After incubation, the digestion solution was added overnight so that the solution completely covered the gel and incubated overnight.

  Next, the digested peptide fragment was extracted with 100 μl of an aqueous solution containing 1 (w / v)% DM, and mixed with a vortex mixer for 1 hour. After extraction, 100 μl of chloroform / methanol (2: 1) was added and mixed, and the organic layer was transferred to another tube. The organic solvent was evaporated and the extracted peptide fragments were dissolved in 100 μl of matrix solution (saturated CHCA / 0.1% TFA in acetonitrile (1: 2)) and spotted on a MALDI plate. Mass spectrometry was performed using a MALDI-TOF mass spectrometer (AXIMA-CFR; manufactured by Shimadzu Corporation) and measured in the positive reflection mode.

  FIG. 8 shows a mass spectrum of PsbQ-His separated by SDS-PAGE using the above method. This gave a clear signal despite reducing the sample used to 25 pmol.

  On the other hand, when only the chloroform / methanol extraction was performed on the trypsin-digested peptide fragment (FIG. 7B) or only the DM extraction was performed, the signal corresponding to the lipid-modified fragment in MALDI-TOF mass spectrometry was Not observed.

  In addition, when trypsin-digested peptide fragments were extracted with acetonitrile / 0.1% TFA (1: 2) and MALDI-TOF mass spectrometry was performed in the same manner, no signals corresponding to the lipid-modified fragments were observed. (Figure 7A). On the other hand, a signal corresponding to T2 ion could be detected, indicating that PsbQ-His was effectively digested by trypsin. Even with 100% acetonitrile containing 0.1% TFA, no signal of the lipid modified fragment was detected.

  Since SDS-PAGE is widely used for proteome analysis, the method of the present invention is particularly useful for proteome analysis of lipid-modified proteins. The method of the present invention is also advantageous in that it does not require special equipment such as HPLC.

FIG. 1A shows the structure of the mature form (lipid-modified form) of PsbQ-His and T ions generated by trypsin digestion. FIG. 1B shows the amino acid sequence and calculated molecular weight of a tryptic digest fragment of PsbQ-His. The mass spectrum of PsbQ-His expressed in E. coli is shown. A signal was observed at 17,918 (m / z). According to the theoretically calculated mass, this signal corresponds to one diacylglycerol and one PsbQ-His modified with one acyl group. The gas chromatogram of the fatty acid methyl ester derived from PsbQ-His expressed in E. coli is shown. Palmitic acid (16: 0) was the most abundant fatty acid. Palmitoleic acid (16: 1), cyclomethylene-heptadecanoic acid (17: 0 cyclo) and vaccenic acid [18: 1 (11)] were also detected. The MALDI-TOF MS spectrum of the tryptic digest fragment of PsbQ-His expressed in E. coli is shown. The tryptic digest fragment of PsbQ-His was analyzed by the commonly used method (A) or the method of the present invention (B). The arrow indicates the signal derived from the N-terminal fragment of PsbQ-His modified with lipid. Figure 2 shows a MALDI-TOF MS spectrum of a lipid-modified N-terminal fragment of PsbQ-His expressed in E. coli. The MS / MS spectrum of the N terminal fragment of PsbQ-His is shown. Several y ions from the N-terminal fragment of PsbQ-His were observed. The MALDI-TOF MS spectrum of PsbQ-His separated by SDS-PAGE is shown. After in-gel digestion, peptide fragments were extracted with acetonitrile / 0.1% TFA (1: 2) (A) or chloroform / methanol (2: 1) (B). The MALDI-TOF MS spectrum of PsbQ-His separated by SDS-PAGE is shown. The trypsin digested fragment was eluted with a solution containing 1% DM and extracted with chloroform / methanol. Proteins corresponding to 1% 100 pmol (A), 25 pmol (B) and 5 pmol (C) were loaded on SDS-PAGE and used for analysis. Signals corresponding to N-terminal fragments modified with lipids are indicated by arrows.

Claims (10)

  A method for extracting a lipid-modified peptide from a sample by extracting the sample containing the lipid-modified peptide and the non-lipid-modified peptide with an organic solvent.   The method according to claim 1, wherein the organic solvent is an organic solvent comprising a low polarity organic solvent and a high polarity organic solvent. (a) Protease treatment of a sample containing lipoprotein
(b) A step of extracting the sample treated with the protease in the step (a) with an organic solvent
(c) A method for analyzing lipoprotein, comprising a step of subjecting the extract obtained in the step (b) to mass spectrometry.   4. The method according to claim 3, wherein in the step (a), a supernatant is obtained by adding a surfactant to a sample containing lipoprotein and centrifuging the sample, and then subjecting the sample to protease treatment.   5. A method according to claim 3 or 4, comprising analyzing the lipid bound to the protein.   The method according to any one of claims 3 to 5, wherein the organic solvent is an organic solvent comprising a low polarity organic solvent and a high polarity organic solvent. (a) Step of subjecting a sample containing lipoprotein to gel electrophoresis
(b) A step of subjecting the gel after electrophoresis to protease treatment
(c) A step of extracting a protease-treated sample with a solution containing a surfactant
(d) A step of further extracting the extract obtained in the step (c) with an organic solvent.
(e) A method for analyzing lipoprotein, comprising a step of subjecting the extract obtained in the step (d) to mass spectrometry.   8. The method of claim 7, wherein the surfactant is a nonionic surfactant.   The method according to claim 7 or 8, wherein the organic solvent is an organic solvent comprising a low polarity organic solvent and a high polarity organic solvent.   10. A method according to any one of claims 7 to 9, comprising analyzing lipids bound to the protein.

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