WO2007108140A1 - Identification method and quantification method for protein sample using electrophoresis and mass spectrometry - Google Patents

Abstract

Disclosed is a method for the analysis of a proteome utilizing electrophoresis which enables the differential display on a single of gel. A method for identification or quantification of a protein sample comprising the steps of (i) providing two protein samples, (ii) modifying the two proteins separately using modification reagents which are only different from each other in the isotope composition, respectively (that is, labeling either of the two protein samples by the isotope labeling method), (iii) mixing the two modified protein samples together, (iv) performing electrophoresis, (v) performing in-gel fragmentation, (vi) concentrating and separating the modified peptides, and (vii) performing mass spectrometry of the resulting peptides.

Description

 Description Identification and quantification of protein samples using electrophoresis and mass spectrometry Technical Field

 The present invention relates to proteomics, that is, a comprehensive analysis method of proteins. More specifically, the present invention relates to a method for identification and quantification of protein using electrophoresis and mass spectrometry using electrophoresis and mass spectrometry.

Background art

Current proteomics (comprehensive analysis of proteins) includes 1) two-dimensional electrophoresis (2D-PAGE) and mass spectrometry (MALD 卜 T0F / MS) PMF analysis, and 2) two-dimensional liquids There are two major flows: analysis using chromatography (2D-HPLG) and mass spectrometry (MALDMAL T0FT0F or ESI-MSMS). The corner of 1) above? In the analysis method, Ettan-DIGE (Deference Gel Electrophoresis) method solved the reproducibility problem of 2D-PAGE, and it became easy to determine the expression level from the relative ratio of fluorescence. This method has the advantage that expression comparison can be visualized. In the analysis method of 2) above, high sensitivity is measured by combining the sensitivity of mass spectrometry and a separation device suitable for a small amount of sample, and various methods are introduced. Several methods using stable isotope labeling are known as quantitative proteome methods that do not use gels. As stable isotope labeling methods, ¹⁶ 0 ₂ Z ¹⁸ 0 ₂ method, IC Examples include AT method, iTRAQ method, NBS method using nitrobenzenesulfenyl chloride (NBS reagent). The NBS method is described in detail in International Publication No. 2004/002950 Pamphlet. Japanese Patent Application Laid-Open No. 2005-189104 describes a method of concentrating and separating a protein peptide modified with an NBS reagent using a carrier having a phenyl group. In Japanese Patent Application Laid-Open No. 2005-326391, a peptide modified with an NBS reagent is designated as α-cyan-3-hydroxygay cinnamate, 3-hydroxy-4-hydroxybenzoate, or 3-hydroxy-1 4 —A mixture of nitrobenzoic acid and α-cyano 4-hydroxygaynamic acid is used as a matrix and measured using a MALD I mass spectrometer to efficiently detect peptides modified with NBS reagent .

 In Japanese Unexamined Patent Publication No. 2006-010672, a mixed sample containing a peptide modified with an NBS reagent and a substance other than that is mixed with a nitrobenzene derivative, or a mixture of a nitrobenzene derivative and a cyano 4-hydroxygaynamic acid. It is used as a matrix, measured using a MALDI mass spectrometer, and peptides modified with NBS reagent are selectively detected from the mixed sample. On the other hand, recently, in the 55th edition of The 3rd JHUP0 Conference, it was reported that proteins modified with NBS reagent were separated by 2D-PAGE.

Disclosure of the invention

Object of the invention

According to the method in which modified proteins are separated by 2D-PAGE using NBS reagent Thus, only about 50% of the proteins identified by the PMF analysis method can be quantitatively analyzed. Also, in this method, for the purpose of performing PMF analysis and quantitative analysis at the same time, 4-one CHCA (α-cyan 4-hydroxycinnamic acid) is used as a matrix. However, as a result of mass spectrometry, in addition to the fragment having an NBS group, a peak derived from the fragment (that is, a peak whose mass number is 32 Da or 16 Da smaller than the peak corresponding to the fragment) is detected. Analysis sensitivity is low. Therefore, this method cannot efficiently detect peptide fragments containing tributophan residues. One of the causes of this problem is that NBS-labeled peptides (that is, NBS group-containing peptides) cannot be concentrated because of gel-based analysis. Since NBS reagent modifies tributophane residues that are abundant in proteins, if NBS group-containing peptides are not concentrated, peptides in MS samples will be labeled with NBS-unlabeled peptides (ie, NBS Group-free peptides). A large amount of NBS group-free peptide causes ion subsidence for the NBS group-containing peptide to be detected by mass spectrometry. For this reason, the detection efficiency of a NB S group containing peptide worsens. Another cause of this problem is that a matrix that is unfavorable for the detection of peptides containing NBS groups is used because both identification and analysis are performed by the PMF method. Accordingly, an object of the present invention is to provide a proteome analysis method using an electrophoresis method, which enables differential display with a single gel. is there. Furthermore, a further object of the present invention is to provide a proteome analysis method using an electrophoresis method, which provides a quantitative reliability and sensitivity.

Summary of the Invention

 The present inventor has found that the object of the present invention can be achieved by removing the NBS group-free peptide and concentrating the NBS group-containing peptide. Furthermore, the present inventor has found that the object of the present invention and the further object can be achieved by using a matrix more suitable for detection of NBS group-containing peptides by mass spectrometry. Based on these findings, the present invention has been completed. The present invention includes the following inventions. The following (1) to (5) are directed to methods for identifying and quantifying protein samples. The following (6) is directed to a separation device that can be usefully used in the methods (1) to (5). The following (7) is directed to a kit that can be usefully used in the methods (1) to (5).

(1) (!) Preparing a protein sample and preparing a protein sample II as a control of the protein sample I;

(ii) preparing two kinds of compounds having different molecular weights by containing isotopes having the same molecular structure and different mass numbers as the modifying reagent, and for the protein sample I, the two kinds While modifying either one of the compounds to obtain a modified protein sample I ′ into which a modifying group derived from any one of the compounds is introduced, Modifying the protein sample II with one of the two compounds and obtaining a modified protein sample I into which a modifying group derived from the other compound is introduced;

 (ii) Mixing the modified protein sample and the modified protein sample I I ′ to obtain a mixed sample for electrophoresis;

 (iv) subjecting the mixed sample for electrophoresis to electrophoresis and spreading the sample into a plurality of protein spots;

 (V) A desired protein spot is cut out from the plurality of protein spots, the cut out protein spot is fragmented in the gel, and the peptide fragment containing the modifying group and other peptide fragments are concentrated. Obtaining a mixed sample for separation;

(vi) Using the carrier having a structure capable of interacting with the modifying group derived from one of the compounds and the modifying group derived from the other compound, from the mixed sample for concentration and separation A step of concentrating and separating the modifying group-containing peptide fragment to obtain a sample for mass spectrometry containing the concentrated peptide fragment containing the modifying group; (vi i) Identifying and quantifying the concentrated and separated modified group-containing peptide fragment,

A method for identifying and quantifying protein samples, comprising: In the above (1), the term “protein” is used to include a peptide having a relatively small molecular weight.

In (1) above, two types of protein samples are prepared in step (i), and the two types of protein samples are separately prepared using two types of modifying reagents that differ only in the isotopic composition in step (ii). Modified (ie, isotope-labeled one of the protein samples by the isotope labeling method), mixed the two kinds of modified protein samples in step (iii), electrophoresed in step (iV), In V) fragmentation in the gel was performed, and in step (vi) the modified peptide Is concentrated and separated, and mass spectrometry is performed in step (vii).

(2) In the step (ii), a compound having an aryl group is used as a modifying reagent, and the modified protein into which the aryl group is introduced as a modifying group and the modified protein 1 are obtained. In the step (vi), The method for identifying and quantifying a protein sample according to (1), wherein a carrier having an aryl group is used as a carrier having a structure capable of interacting with the modifying group.

(3) The protein sample identification and quantification method according to (2), wherein the aryl group of the carrier is a phenyl group.

(4) The compound having an aryl group as the modifying reagent is 2-nitro [ ¹³ C ₆ ] benzenesulfuryl chloride and 2-nitro [ ¹² C ₆ ] benzenesulfuryl chloride. 2) The protein sample identification and quantification method according to (3).

(5) The protein sample identification and quantification method according to any one of (1) to (4), wherein in the step (vi), the carrier is used by being filled or fixed in a pipette tip. (6) In the step (vii), as a matrix,

 3-hydroxy-1,4,12-trobenzoic acid and / or mono-cyano 3-hydroxycinnamic acid,

 1-Ciano 1-hydroxycinnamic acid,

The protein sample identification and quantification method according to any one of (1) to (5), wherein the mixture is used. (7) A tip for concentration and separation, in which a pipette tip is filled or fixed with a carrier having a phenyl group.

(8) A tip for concentration / separation in which a pipette tip is filled or fixed with a carrier having a phenyl group;

2-nitro ^[13 C _6] benzene sulphates enyl chloride Li de and 2 two Toro ^[12 C _6] and base emissions Zen sulphates enyl chloride Li de,

Including kit. According to the present invention, there is provided a proteome analysis method using electrophoresis, which enables a differential display with a single gel. Furthermore, according to the present invention, there is provided a proteome analysis method using an electrophoresis method, and an analysis method with high reliability and sensitivity of quantification.

Brief Description of Drawings

 Figure 1 shows the results obtained by subjecting sample 1 obtained by electrophoresis and in-gel digestion to a protein sample labeled with an isotope labeling method using NBS reagent, and desalting purification using Ziptip. This is the MA LD I—TOF MS spectrum of the sample for mass spectrometry.

 Figure 2 shows a sample sample obtained by electrophoresis and in-gel digestion of a protein sample labeled by the isotope labeling method using NBS reagent. 2 is a MA LD I-TOF MS spectrum of a sample for mass spectrometry obtained by concentrating and separating an NBS group-containing peptide fragment by ιρ.

Figure 3 shows protein samples labeled with the isotope labeling method using NBS reagent. The sample is obtained using the MA LD I-TOF MS spectrum of the sample for mass spectrometry obtained by desalting and purification using Ziptip on sample 2 obtained by electrophoresis and in-gel digestion.

 Figure 4 shows sample 2 obtained by electrophoresis and in-gel digestion of a protein sample labeled with an isotope labeling method using an NBS reagent. Desalting and purification using Ziptip and NBS group using Phenyltip 2 is a MALD I-TOF MS spectrum of a sample for mass spectrometry obtained by concentrating and separating a peptide fragment contained therein.

 Figure 5 shows Pheny's analysis of NBS group-containing peptide fragments by ip against sample 2 obtained by electrophoresis and in-gel digestion of a protein sample labeled by the isotope labeling method using NBS reagent. It is a MA LD I-TOF MSZMS spectrum of the sample for mass spectrometry obtained by performing concentration separation.

 Figure 6 is obtained by desalting and purifying Sample 3 obtained by electrophoresis and in-gel digestion of a protein sample labeled with an isotope labeling method using NBS reagent. This is a MA LD I-TO F MS spectrum of a sample for mass spectrometry.

 Figure 7 shows sample 3 obtained by electrophoresis and in-gel digestion of a protein sample labeled by the isotope labeling method using NBS reagent, and desalted by Ziptip and NBS group by Phenyltip. It is a MALD I-TOF MS spectrum of a sample for mass spectrometry obtained by concentration separation of peptide fragments.

BEST MODE FOR CARRYING OUT THE INVENTION

<Protein identification and quantification method>

The present invention provides protein identification and quantification methods. Identification and quantification method of the present invention In the step (i) of preparing two kinds of protein samples, two kinds of proteins are modified separately using a modification reagent that differs only in the isotopic composition (that is, one of the two isotopes is labeled by the isotope labeling method). Labeling a protein sample) step (ii), mixing two modified protein samples (iii), performing electrophoresis (iv), performing gel fragmentation, and step (V) A step (vi) for concentrating and separating the modified peptide, and a step (vi) for performing mass spectrometry.

(i: Preparation of protein sample)

 First, prepare two types of protein samples for protein identification and quantification. Examples of the two kinds of protein samples include those having the following relationship.

 For example, if one protein is a protein sample from one sample and the other protein is a protein sample from another sample; one protein is the protein sample to be analyzed and the other protein sample is When it is a control protein for said one protein; when one protein sample is a protein sample extracted from a pathologic sample and the other protein sample is a protein sample extracted from a normal sample, etc. .

It is preferable to prepare the same amount of the two types of proteins used in the present invention so as to facilitate analysis in later mass spectrometry. The protein sample may be collected and / or extracted from an individual and then solubilized. The solubilization method is not particularly limited. For example, a protein can be solubilized using a surfactant such as sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride, etc. as a denaturing agent. The concentration of denaturing agent is not particularly limited, so that protein samples can be solubilized and denatured. A person skilled in the art may determine as appropriate in consideration of the type of sample and other conditions. The reaction conditions may be appropriately determined by those skilled in the art in consideration of the modifying agent to be used regardless of normal temperature modification or heat modification.

(i i) Modification of protein sample

In this process, one of the two proteins isotope-labeled by the so-called isotope labeling method. At this time, an appropriate group is introduced as a modifying group into both of the two types of proteins so that the molecule to be detected in the present invention can be efficiently concentrated and separated in the subsequent step (vi). In this step, a specific structure in the protein is used as the modification target. The modifying reagent is particularly limited to a compound having a group capable of selectively modifying the specific structure and capable of interacting with the carrier used in the subsequent step (vi). It can be used without using it. In the present invention, for example, a triftophan residue in a protein may be used as a modified target. In addition, the modifying reagent in the present invention uses a combination of two compounds having the same molecular structure but having different molecular weights by containing isotopes having different mass numbers. Of these, compounds with higher molecular weights are used as heavy reagents, and reagents with lower molecular weights are used as light reagents. More specifically, a compound in which at least one element constituting the modifying reagent molecule is labeled with a stable isotope has the same structure, but the aforementioned element is a stable isotope. Use in combination with a compound labeled with another stable isotope. Then, the compound labeled with a stable isotope having a large mass number is used as a heavier reagent, and the other compound is used as a light reagent. There may be a plurality of elements labeled with stable isotopes. Specifically, when protein samples I and II are prepared, protein sample I is modified with one of a heavy reagent and a light reagent to obtain a modified protein sample, and protein sample II is prepared with a heavy reagent and a light reagent. Modify with either one of the reagents to obtain modified protein II '. When a tribtophan residue is used as a modification target,

1: (R, represents an organic group, X represents a leaving group) or a compound represented by the general formula 2: R ₂ -X ₂ (R ₂ represents a substituted aralkyl group. X ₂ represents a leaving group), and the like. By using such a modifying reagent, one modifying group R 1 S or 1 R ₂ is introduced as a modifying group into the protein. As the compound represented by the above general formula 1, that is, a sulfinyl compound, aryl sulfenyl halide in which in the above general formula 1 is substituted is a aryl group. Furthermore, as the arylsulfenyl halide, 2-nitrobenzenesulfenyl chloride (NBSG NBS reagent) is preferable.

When 2-nitrobenzenesulfuryl chloride is used, as shown in the following formula, 2-nitro [ ¹³ C ₆ ] benzenesulfuryl chloride (NBSGI heavy) as a heavy reagent and 2-2 It is preferable to use in combination with Toro [ ¹² C ₆ ] benzenesulfuryl chloride (NBSGI light).

NBSCI heavy reagent and NBSCI light reagents, both manufactured by Shimadzu Corporation ^{13 G NBS (R) Stable Isotope} Label ing Kit - sold housed in N. Trobenzenesulfenyl chloride (NBSC I)

 Heavy reagents Light reagents In addition, sulf-L nyl compounds as modifying reagents for tributophan residues in proteins and peptides are described in detail in International Publication No. 2004/002950 / Nflet. As the compound represented by the general formula 2, benzyl halide is preferable. Furthermore, examples of the benzyl halide include 2-hydroxy-5-nitrobenzyl bromide (2-hydroxy-5-nitrobenzyl bromide).

When 2-hydroxy-5-nitrobenzylbutamide is used, 2-hydroxy-5-nitro [ ¹³ C _S ] benzyl bromide as a heavy reagent and 2-hydroxy-5- Nitro [ ¹² C ₆ ] benzylbutamide is preferably used in combination.

2-Hydroxy-5-nitrobenzene

Heavy reagent light reagent As for the benzyl halide compound as a modification reagent for the tributophane residue in the protein 'peptide, see Horton, HR and Koshland, D.E, Jr. (1972), Modification of proteins with actibenzylyl amide, Methods in It is described in detail in ENZYM0L0GY, 25, 468-482, etc. The modifying reagent is not limited to the compounds listed above, and a person skilled in the art appropriately selects a compound having the ability to modify a specific structure in the protein and is particularly limited as long as it is combined with a heavy reagent and a light reagent. Not.

(iii: Preparation of mixed sample for electrophoresis)

 The two modified protein samples I ′ and II ′ obtained by the above modification process are mixed together. In the mixed sample for electrophoresis obtained in this step, the mixed modified protein sample may be appropriately subjected to a treatment such as a reductive alkylation treatment. The reductive alkylation treatment can be performed by a usual method. In addition, this process may be performed in the following step (V) instead of being performed in this step. The mixed sample for electrophoresis obtained in this step may use a normal buffer known to those skilled in the art, including Tris-HG, SDS, -mercaptoethanol, glycerol, and the like.

(iv: electrophoresis)

The electrophoresis sample obtained by the above process is subjected to electrophoresis. As a specific method of electrophoretic movement, a method known to those skilled in the art may be used. For example, isoelectric focusing, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or By performing gel electrophoresis such as two-dimensional electrophoresis (2D-PAGE) combining them, it is developed into multiple protein spots. The detection of the protein after the electrophoresis is preferably performed by Coomassie prioriant blue staining and fluorescence detection.

(V: gel fragmentation)

 A desired protein spot is cut out from the plurality of protein spots obtained in the above process. In-gel fragmentation is performed on the excised protein spots. The fragmentation method may be enzymatic fragmentation or chemical fragmentation. Preferably, digestion using an enzyme such as trypsin is performed. By performing in-gel fragmentation, the desired protein cleaved is fragmented into a peptide fragment containing a modifying group and other peptide fragments. For example, when an N B S reagent is used as a modifying reagent in the above-described step (U), this step results in fragmentation into a peptide fragment having an N B S group and a peptide fragment having no N B S group. The mixture of the thus obtained modifying group-containing peptide fragment and its ^ (ya's peptide fragment) is subjected to an appropriate treatment if necessary, and becomes a mixed sample for concentration and separation described later. Examples of the treatment to be performed include reduction, alkylation, and desalting treatment, etc. This desalting treatment can be performed using a desalting instrument such as Ziptip (manufactured by Millipore).

(vi: concentrated separation)

In this step, the modified group-containing peptide fragment is concentrated and separated from the obtained mixed sample for concentration and separation (mixture of modified group-containing peptide fragments and other peptide fragments). Introducing a concentration / separation step in the present invention is preferable because it enables highly efficient detection of a desired modified group-containing peptide fragment. Specifically, peptide fragments containing tributophane with a low content in the protein are efficiently concentrated and separated, so that the peptide fragments containing tributophane are detected with higher sensitivity by mass spectrometry than when they are not concentrated and separated. The For this reason, protein quantification is also improved. As the concentration / separation method, it is preferable to use a separation instrument in which a small instrument such as a pipette tip is filled or fixed with a carrier. In the present invention, since the gel-based concentration separation is performed, the sample scale is extremely small. Using a separation device in which a carrier is packed or fixed in a small device such as a pipette tip is very suitable for concentration separation on such a small sample scale. Moreover, it is preferable to use such an instrument because it can reduce complicated operations and can be applied to an automated apparatus. As the carrier used in the concentration separation, a carrier having a group capable of interacting with the modifying group introduced in the modifying step (U) is used. The interaction here is an action based on affinity such as van der Waals interaction, 7Γ-7Γ electron interaction, hydrophilic interaction and hydrophobic interaction. In addition, as the carrier having a group capable of interacting with the modifying group, ODS, silica gel, cephadex, and the like may be used. In the present invention, as described above, since a separation instrument having a small standard is used, it is preferable that an ODS-based carrier is used from the viewpoint of workability in the production of the separation instrument. Furthermore, it is preferable to use a monolith type carrier with high decomposition performance as the ODS-based carrier. The monolith type ODS column differs from the widely used packed column in that the framework on the 3D network and its voids (ie, the flow path) are integrated. The carrier and the column are in the form of a body and do not require frit. For example, arylsulfenyl which is a preferred form of the compound represented by the above-described modification step (wherein i represents general formula 1: F ^ —S—X, represents an organic group and X represents a leaving group) When a halide is used, an R, 1 S— group (in this case is an aryl group) is introduced as a modifying group. In the modification step (ii), R ₂ _X ₂ (R ₂ represents an aralkyl group, X ₂ represents a leaving group), the R ₂ — group is introduced as a modifying group When a π-electron group is introduced, such as a modifying group having an aryl group. In this step, a carrier having an aryl group is preferably used as the carrier, and a carrier having a phenyl group is preferable as the carrier having an aryl group, in which case the peptide fragment to be concentrated and separated from the carrier is used. 7Γ- π-electron interaction, and sparse Specific selectivity due to aqueous interaction enables specific enrichment separation Hi-trap phenyl FF, Hi-Trap phenyl HP, Phenyl Sepharose 6 Fast Flow, Phenyl Sepharose Hi h Performance ^ (above, manufactured by Amersham Biosciences), YMC * GEL Ph (manufactured by YMC Co., Ltd.), and other carriers used in the vinyl column.

In the present specification, a separation device in which a carrier having a phenyl group is filled in a pipette tip may be referred to as a Phenyltip. Further details of Phenyltip will be described in “Concentration separation chip and kit containing it” described later. In the present invention, a combination using an NBS reagent as a modifying reagent in the step (ii) and using a carrier having a phenyl group in the step (vi) is particularly preferable. This place The NBS-modified peptide in the NBS-modified peptide due to the interaction of the 7Γ electron of the triphphane in the peptide with the NBS-modified group and the π-electron of the phenyl group in the carrier. Exhibits excellent holding ability against Using such a carrier, only the desired peptide fragment having a modifying group is adsorbed from the peptide fragment mixture. Thereafter, desalting treatment is preferably performed using a mixed aqueous solution of acetonitrile and TFTF (trifluoroacetic acid). In this mixed solution, the concentration of acetonitrile is preferably about 5 to 25% by volume. More specifically, 15% acetonitrile and 0.1% TFA aqueous solution (v / v / v) may be used. By performing such a desalting treatment, chemical species other than the modifying group-containing peptide can be positively removed. The adsorbed modification group-containing peptide fragment is eluted with an appropriate solution and then subjected to an appropriate treatment as necessary to become a sample for the next mass spectrometry. When performing MALDI mass spectrometry in the mass spectrometry step (vii) described later, a matrix may be included in the sample for mass spectrometry. The matrix will be described in detail in step (vii) below. That is, the elution solution can be appropriately determined by those skilled in the art depending on the type of the modified modifying group-containing peptide fragment. For example, when a hydrophobic group such as an NBS group is introduced as a modifying group in the step (ii) and MALDI mass spectrometry is performed in the step (vii) described later, it may be eluted using a matrix solution described later. The matrix (described later) preferably used in the present invention is very compatible with an NBS group-containing peptide, particularly when an NBS group is introduced as a modifying group. Therefore, elution with a preferred matrix solution improves workability for subsequent mass spectrometry measurements. In addition, there is an advantage that a desired peptide fragment can be eluted more efficiently. Note that, for example, a fractionation process is performed as an appropriate process performed as necessary.

 The method for desalting can be carried out by methods known to those skilled in the art. Examples of the method for performing fractionation include a method using a system using a reverse phase column and HPLC.

(Vi mass spectrometry)

 The sample for mass spectrometry obtained in the above step is subjected to mass spectrometry. A MALDI mass spectrometer can be used for the measurement in this step. In this case, a MALDI-T0F type mass spectrometer (eg Shimadzu Corporation / Craitos AXIMA- GFR plus) or a MAID 卜 IT-T0F type mass spectrometer (eg Shimadzu Corporation / Crates AXIMA-QIT) is used. Used.

When a MALDI mass spectrometer is used, the matrix used for the sample for mass spectrometry is not particularly limited. For example, DHB (2,5-dihydroxybenzoic acid; 2, 5-dihydroxybenzoic acid), 4-CHCA (Hycyan 4-hydroxycinnamic acid; -cyano-4-hydroxycinnamic acid), 3-CHCA (—cyano 3-hydroxy Cinnamic acid (a-cyano-3-hydroxycinnamic acid), 3H4NBA (3-hydroxy-4-nitrobenzoic acid), etc. can be used as a matrix. When using a MALD 卜 T0F mass spectrometer, 4-GHGA should be used as the matrix. On the other hand, when a MAIDI-IT-T0F mass spectrometer is used, a mixture of 3H4MBA and / or 3-GHGA and 4-GHGA may be used as a matrix. 3-CHCA and 3H4MBA are preferably used when a hydrophobic π-electron group such as an NBS group is introduced into the protein in the step (i ί). By using these matrices, specific interaction of the desired peptide fragment can be achieved by the affinity resulting from the hydrophobic interaction and the π-electron interaction, and the self-disintegration of the peptide fragment (especially , And the elimination of nitro group oxygen in the NBS group.

 Further, a mixed matrix obtained by mixing 3-GHGA or 3Η4Η with 4-CHGA is particularly preferably used when a hydrophobic group such as an NBS group is introduced into a protein in the step (ii). Such a mixed matrix is an excellent matrix having the above-mentioned specific ionization ability and self-decay suppression ability as well as high-sensitivity measurement ability. For this reason, the quantitative reliability of the method of the present invention is very high. The compound used as a matrix can be appropriately determined by those skilled in the art for the purpose of being used as a matrix for mass spectrometry. For example, these compounds can be used as a solution having a concentration of 1 mg / mI to saturation.

As a solvent used for the preparation of such a solution, it is preferable to use an aqueous solution of acetonitrile, an aqueous solution of trifluoroacetic acid (TFA), or an aqueous solution of acetonitrile-trifluoroacetic acid (TFA). When using an acetonitrile solution or an acetonitrile-TFA aqueous solution, the concentration of the acetonitrile is not particularly limited, but 90% or less, preferably about 7 O v / v% can be used. When using a TFA aqueous solution or acetonitriru TFA aqueous solution, the concentration of TFA is not particularly limited, but it may be 1 ν / νο / ₀ or less, preferably about 0.1 ν / ν%.

By dissolving in such a solvent, 1 mg / ml to saturated concentration for 4-CHGA, preferably 5 to 8 mg / m I; 1 mg / m I to saturated for 3-CHGA concentration, In the case of 5 to 8 mg / m I; 3H4NBA, it can be used as a matrix solution of 1 mg / m I to a saturated concentration, preferably 5 to 8 mg / m I. When used as a mixed matrix, the matrix solution is preferably used in a volume ratio of 1:10 to 10 _: 1, more preferably 1 _: 3 to 3: 1, for example, 1: 1. As described above, such a matrix solution can be used for elution of the peptide fragment containing the modified group separated in the step (vi). When hydrophobicity such as NBS group is introduced into the protein in the step (M), the use of such an elution solution means that the modifying group-containing peptide fragment to be eluted in the step (vi) 7Γ- 兀, which is thought to work with the matrix contained in the solution, has the advantage of efficient elution of the peptide fragment containing the modifying group due to the affinity resulting from electron interaction and hydrophobic interaction. is there. In the present invention, the modification group-containing peptide is detected as a pair peak by MS measurement, and the protein can be quantified from the intensity ratio of two peaks constituting the pair peak. On the other hand, protein identification may be performed by PMF analysis or MSZMS analysis. When performing identification and quantification using a single mass spectrometer, identification may be performed by PMF analysis and quantification by MS analysis. In this case, the sample after gel fragmentation is desalted using Ziptip to prepare a sample for mass spectrometry for identification by PMF analysis. Samples for mass spectrometry can be prepared for quantification by MS analysis by concentrating and separating the modifying group-containing peptide fragments using Phenyltip or the like. PMF solution For mass spectrometry samples for analysis, 4-CHCA is preferably used as the matrix. For the sample for mass spectrometry for MS analysis, it is preferable to use a mixture of 3 H4 NBA and / or 3-0! To 10 and 4-01 "10 8 as a matrix. Samples for mass spectrometry can be dropped on a single MS plate and used for mass spectrometry measurement, ie, this method requires the preparation of two types of mass spectrometry samples. Identification and quantification can be performed with this mass spectrometer.

When identification and quantification are performed using two mass spectrometers, identification can be performed by MS / MS analysis and quantification can be performed by MS analysis. In this case, 'Pheny can concentrate and separate the modifying group-containing peptide fragment using ip or the like on the sample after gel fragmentation to prepare a sample for mass spectrometry. Before the concentration and separation treatment, desalting treatment may be performed using Ziptip or the like. It is preferable to use a mixture of 3 H 4 NBA and / or 3-CH CA and 4-CH CA as a matrix for the sample for mass spectrometry. The prepared sample for mass spectrometry can be used for MS measurement for quantification and MSZMS measurement for identification. In other words, with this method, identification and quantification can be performed simply by preparing a single sample for mass spectrometry, and in an environment where not only an MS measurement mass spectrometer but also an MS ZMS measurement mass spectrometer can be used. This is a very useful method. In any of the above cases, it is possible to perform a differential display with a single gel. In this respect, the present invention can be said to be a very useful method. <Concentration separation chip and kit containing it>

 The present invention also provides a chip for concentration and separation. The concentration and separation chip of the present invention is an instrument that can be usefully used in the protein identification and quantification method of the present invention. More specifically, a chip for concentration and separation that is preferably used in the preferred form of the above-described protein identification and quantification method, that is, the form using the N B S reagent.

(Pheny is an ip phenyl chip), and among those described in the above step (vi), a carrier having a phenyl group is filled or fixed. Hereinafter, more specific specifications of the concentration / separation apparatus (Pheny is ip) of the present invention will be described. The instrument in which the carrier is filled or fixed is a pipette tip. Examples of the material of the instrument include polypropylene resin, polyethylene resin, and fluororesin (such as PTEE, PTFE, and ET FE) that are used in commercially available pipette tips. The dimensions of the device can be, for example, a volume of 5 to 1 000 L and a length of 3 to 5 cm, for example. The carrier is as described in the above step (νί) in the protein identification and quantification method of the present invention. A particularly preferred form of the concentration and separation chip (phenyl chip) is one in which a carrier having a filter shape in which the surface of monolithic silica is modified with a phenyl group is fixed to a pipette chip. The use conditions of concentration and separation chip of the present invention, the temperature can be 15 to 40 ° C, _P H 7 or less. Preferably, it is used by being attached to an apparatus having a pump function capable of sucking and discharging liquid, such as a micropipette, and is preferably used by being incorporated in an automation device. As an automation device, it can be used for equipment such as Xcise (manufactured by Shimadzu Corporation). For adsorption of samples for concentration and separation For this purpose, suction and discharge can be performed through the carrier in the chip. When the adsorbed molecules are eluted, the elution solution can be sucked up and discharged through the carrier in the chip. By using the chip for concentration and separation of the present invention, a sample on a very small scale (that is, a sample containing molecules at femtomole to picomolar level) such as a sample cut out from an electrophoresis gel and subjected to fragmentation in the gel. Even in such a case, the concentration and separation of the desired molecule can be performed simply and with high reproducibility and recovery rate. The present invention also provides a kit including the concentration and separation chip. The kit of the present invention is a kit that can be usefully used in the identification and quantification method of the protein of the present invention. More specifically, the kit is preferably used in the preferred form of the above-described protein identification and quantification method, that is, in the form using the NBS reagent. In addition to the concentration separation chip described above, the NBS reagent, ie, 2 -Nitro [ ¹³ C ₆ ] benzenesulfuryl chloride and 2-nitro [ ¹² C ₆ ] benzenesulfuryl chloride are included as kit contents. In addition to this, a TFA aqueous solution (for example, about 1 V / V <½), acetonitrile, or the like may be included in the kit contents.

Example

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. <Preparation of sample 1, sample 2 and sample 3>

 (i) 0.1 g of cells of a wild strain of E. coli and a cra / mg-deficient strain were collected, suspended in 2 ml of 8M urea aqueous solution, and subjected to ultrasonic disruption.

(M) Stable isotope labeling was performed on each ultrasonic disruption solution. Specifically, an acetic acid (1 OOjUL) solution of ¹² C NBS reagent (I mg) was added to a wild strain of ultrasonic disruption solution 500 /. On the other hand, ¹³ C NBS reagent (1 mg) in acetic acid (1 00 jU L) was added to 50 Oju of the ultrasonically disrupted cra / mg-deficient strain. Both samples were reacted for 3 hours at room temperature.

 (iii) After completion of the reaction, both reaction solutions were mixed, and excess NBS reagent was removed using an LH-20 column (desalting column). Thereafter, reductive alkylation was performed using chloroacetamide and DTT. In this way, a sample for electrophoresis was obtained.

 (iv) The obtained sample for electrophoresis was subjected to two-dimensional electrophoresis. Specifically, IPG Ready strip (pH 5-8) gel is used (or pH 4-7 can be used), and isoelectric focusing is performed as the first dimension. SDS-PAGE was performed. After electrophoresis, G 250 staining was performed to detect protein spots.

 (V) The detected spot was cut out. Specifically, three spots were cut out, and each spot was digested with enzyme in the gel using trypsin. The digests obtained were designated as Sample 1, Sample 2, and Sample 3, respectively.

<Experimental example 1>

 Sample 1 is Pheny's sample that does not perform concentration separation by ip (Sample 1 1 a),

The sample was divided into the sample to be concentrated and separated by Phenyltip (Sample 1-b) and analyzed by the following method. [Sample 1 a]

 Sample 1—a was desalted and purified using Ziptip. After that, MS was prepared using a matrix solution prepared with 4 mg CH CA (—Cyanol 4-hydroxycinnamic acid) in a concentration of 5 mgZm I in 0% acetonitrile — 0 · 1% TFA aqueous solution (v / v / v). The sample was eluted in a single volume and used as a sample for mass spectrometry. About this sample for mass spectrometry, MS measurement was performed with a MALDI-TO-F mass spectrometer (AXIMA-GFR, manufactured by Shimadzu Corporation), and PMF analysis was performed.

As a result of PMF analysis, identification with a high score (1 47) was possible. For example, as an NBS group-containing peptide fragment, a ¹³ CN BS group-containing peptide fragment having the sequence of KQYPIVSIEDGLDESDWDGFAYQTKV (SEQ ID NO: 1) was detected.

However, MA LDI -TO F MS scan Bae spectrum (Figure 1) shows, on the one hand the peptide fragment having ¹³ CNBS group ions being barely detectable, from peptide fragments having the ¹³ CN BS group 1 6 Da and 32 Da mass numbers are also detected. In addition, in FIG. 1, in the range of m / z 2898-2952, ions of a peptide fragment having a ¹³ CNBS group, and ions having ¹⁶ Da and 32 Da mass numbers smaller than the fragment are detected. It is enlarged and displayed separately. These ions with lower mass numbers are generated by the loss of one or two nitro group oxygens in the peptide fragment with ¹³ CN BS groups, respectively. Thus, “dividing one peak of a peptide fragment having a CNBS group into two peaks means an unfavorable result from the viewpoint of analysis sensitivity.

[Sample 1—b]

Sample 1-b was desalted and purified using Ziptip. After that, Phenyltip (filter type silica monolith is chemically bonded to phenyl group, and 10 ~ 1 volume pipette The NBS group-containing peptide fragments were concentrated and separated using a fixed to a chip and obtained by custom order from Kyoto Monotech Co., Ltd. In concentrated separation, chemical species other than NBS group-containing peptide fragments are actively removed by desalting with 0.1% aqueous TFA (0.1% TFA) (v / v / v). did. After that, 4-CHCA (monocyan-4-hydroxycinnamic acid) was prepared at a concentration of 5 mgZm I in 70% acetonitrile and 0.1% TFA aqueous solution (v / v / v). Prepare a 1: 1 (volume ratio) mixed matrix solution of 3H4NBA (3-hydroxy-4-nitrobenzoic acid) at a concentration of 5 mgZ m I in the same aqueous solution and mix the NBS group-containing peptide fragments with MS. The sample was eluted on a plate and used as a sample for mass spectrometry. About this sample for mass spectrometry, MS measurement was performed with a MALD I-TOF type mass spectrometer (AXIMA-CFR, manufactured by Shimadzu Corporation). Figure 2 shows the obtained MALD I-T OF MS spectrum. In FIG. 2, the range near m / z 2900-2960 where ions of peptide fragments having ¹³ CNBS groups are detected is shown separately enlarged. As shown in the MA LDI-TO FMS spectrum in Fig. 2, the only peptide fragment ion with ¹³ CNBS groups was detected and generated from the peptide fragment as detected in Sample 1-a above. One 16 Da and one 32 Da peaks were not detected. Therefore, it can be said that favorable results were obtained from the viewpoint of analysis sensitivity. Thus, since it is not necessary to perform PMF analysis on sample 1b, it was possible to perform highly sensitive analysis by using a mixed matrix. By the way, when identified by PMF analysis, a ¹³ CNBS group-containing peptide fragment having the sequence of KQYPIVSIEDGLDESDDGFAYQTKV (SEQ ID NO: 1) was detected as an NBS group-containing peptide fragment, but the score was 77, sample 1—a It was lower than the score. As can be seen from the above results, the sample 1 a has a high identification score, but the analysis caused by the 16 Da and 32 Da fragments generated undesirably from the peptide fragments to be detected. The sensitivity was lowered. This is probably because one CHCA was used as a matrix for the purpose of PMF analysis. Sample 1b could be analyzed with high sensitivity without producing such 116 Da and 32 Da fragments. One reason for this is that it is not necessary to perform PMF analysis, so it seems that the use of a mixed matrix was successful.

<Experimental example 2>

 Sample 2 is a sample that is not concentrated and separated by Phenyltip (Sample 2-a),

It was divided into samples (sample 2-b and sample 2-c) to be concentrated and separated by Phenyltip and analyzed by the following method.

[Sample 2— a]

 Sample 2—a was subjected to the same treatment as Sample 1—a.

As a result of PMF analysis, it was possible to identify with high scores (1 1 2, 1 1 0). For example, as an NBS group-containing peptide fragment, a ¹³ CNBS group-containing peptide fragment having the sequence of KKLLPWIDGLLDAGEKH (SEQ ID NO: 2) was detected. In the MALD I-TO FMS spectrum (Fig. 3), only one peptide fragment with an NBS group was detected. In FIG. 3, the range around m / z 2900-2960 where ions of peptide fragments having ¹³ CNBS groups are detected is shown separately enlarged. However, it is known that there are actually three types of peptide fragments having NBS groups in Sample 2. (This is also shown in the analysis results of Sample 2-b described later.) Nevertheless, the fact that only one peak of the NBS group-containing peptide fragment could be detected was the viewpoint of analysis sensitivity. This shows that the result was not preferable.

[Sample 2-b]

 Sample 2-b was subjected to the same treatment as sample 1-b.

In the obtained MA LDI-TO FMS spectrum (Fig. 4), it was possible to clearly detect three paired peaks indicating the presence of peptide fragments having NBS groups. In FIG. 4, ions of peptide fragments having NBS groups are detected, in the range of m / z 1418-1436, in the range of m / z 1692-1714, and in the range of m / z 1814-1842. Each range is enlarged separately. In FIG. 4, the intensity ratio of the two peaks constituting the pair peak was the same for all three pair peaks. From this, it can be seen that highly sensitive analysis can be performed, and results with high reliability of quantification were obtained. On the other hand, as a result of identification by PMF analysis, all NBS group-containing peptide fragments; ¹³ CN BS group-containing peptide fragments having the sequence of KKLLPWIDGLLDAGEKH (SEQ ID NO: 2) and ¹² CN BS group-containing peptide fragments, KLLPWIDGLLDAGEKH (sequence) No. 3) ¹³ CNBS group-containing peptide fragment and ¹² CNBS group-containing peptide fragment, MN1DTDTQWATWEGVLNYYKA (SEQ ID NO: 4) ¹³ CNBS group-containing peptide fragment, KYYDPRVWLRA (SEQ ID NO: 5) sequence ¹³ CNBS group-containing peptide fragments and ¹² CNBS group-containing peptide fragments were detected, but the scores were 28 and 26, which were lower than the scores in sample 2-a. However, as shown in the results of Sample 2-c described later, the peak of the NBS group-containing peptide fragment and the peak of the NBS group-free peptide fragment in the MS spectrum of Fig. 4 were used as precursor ions, respectively. If MSMS measurement is performed, it is possible to perform identification with higher reliability.

[Sample 2-c]

 Sample 2—c was treated in the same manner as Sample 1b above, except that desalting and purification using Z ί pt ip was not performed.

 In MA LD I—TOF MS spectrum (not shown), as in FIG. 4, three paired peaks indicating N B S group-containing peptide fragments were detected. On the other hand, when PMF analysis was performed,

A peptide fragment containing the sequence of ¹³ CNBS group containing the sequence of KKLLPWIDGLLDAGEKH (SEQ ID NO: 2) and a peptide fragment of SKIFDFVKPGVITGDDVQKV (SEQ ID NO: 6) as an NBS group-free peptide fragment was detected, but the score was 52 It was lower than the score in Sample 2-a. However, the MSZMS measurement using the peak of the NBS group-containing peptide fragment having the sequence of SEQ ID NO: 2 and the peak of the NBS group-free peptide fragment having the sequence of SEQ ID NO: 6 as precursor ions, respectively, was performed using the mass spectrometer AXIMA -Identification was possible by using QIT (manufactured by Shimadzu Corporation). Figure 5 (a) shows the MSZMS spectrum using the NBS group-containing peptide fragment as a precursor ion, and Fig. 5 (b) shows the MS spectrum using the NBS group-free peptide fragment as a precursor ion. ). From this, it can be seen that the Zipt ip treatment does not necessarily have to be performed if sufficient desalting with Phenyltip and concentration separation of the NBS group-containing peptide are performed. As can be seen from the above results, in sample 2-a, the NBS group-containing peptide fragment could not be specifically detected while the identification score was high. This is probably due to the fact that only desalting with Ziptip was performed as a purification process. In Sample 2-b, all NBS group-containing peptide fragments could be detected, and by detecting multiple paired peaks, a highly reliable result of quantification was obtained. This is thought to be because one of the reasons was the concentration and separation by Phenyltip as a purification process. In addition, Sample 2c also gave results with high quantitative reliability. Furthermore, although the identification score by the PMF analysis was low, the identification by the MSZMS analysis could be performed instead. In other words, concentration separation using a vinyl column and the use of a mixed matrix lead to a decrease in the identification score of PMF analysis, but instead, MSZMS analysis can be used to perform more reliable identification. It was shown that. ,

<Experimental example 3>

Sample 3 is a sample that Pheny does not perform concentration separation with ip (Sample 3- _a ),

The sample was divided into the sample to be concentrated and separated by Phenyltip (Sample 3-b) and analyzed by the following method.

[Sample 3—a]

 Sample 3—a was subjected to the same treatment as Sample 1-a.

As a result of PMF analysis, identification with a high score (1 97) was possible. However, NBS group-containing peptide fragments were not detected. NBS group-containing peptide fragments were not detected, as shown in MALD I-TOFMS spectrum (Fig. 6). In Fig. 6, the range of m / z 1370-1386 is enlarged and displayed separately. However, it has been found that in Sample 3, there is actually one type of NBS group-containing peptide fragment. (This is also shown in the analysis results of Sample 3-b described later.) Nevertheless, the failure to detect the peak of the NBS group-containing peptide fragment is not preferable from the viewpoint of analysis sensitivity. It turns out that it was a good result. [Sample 3-b]

 Sample 3—b was treated in the same way as Sample 1—b.

In the obtained MA LDI-TO FMS spectrum (Fig. 7), it was possible to clearly detect one pair peak indicating the presence of a peptide fragment having an NBS group. In FIG. 7, the range of m / z 1369-1386 where ions of peptide fragments having an NBS group are detected is shown separately enlarged. This indicates that high-sensitivity analysis can be performed, and results with high quantitative reliability were obtained. On the other hand, as a result of identification by PMF analysis, ¹ ′ ³ CNBS group-containing peptide fragments and ¹² CNBS group-containing peptide fragments having the sequence of KALEGDAEWEAKI (SEQ ID NO: 7) were detected as NBS group-containing peptide fragments. Was 31, which was lower than the score in Sample 3-a. However, as shown in the result of Sample 2-c above, the peak of the NBS group-containing peptide fragment and the NBS group-free peptide fragment in the MS spectrum of FIG. If MS ZM S measurement is performed with each peak as a precursor ion, more reliable identification can be performed. As shown by the above results, in Sample 3-a, the identification score was high, but NBS group-containing peptide fragments could not be detected. This is thought to be due to the fact that only desalting with Ziptip was performed as a purification process. In Sample 3-b, the NBS group-containing peptide fragment could be detected as a pair peak, resulting in high quantitative reliability. This is probably due to the fact that Pheny ltip further concentrated and separated as a purification process. Actually, rNumber of mass va l ues matchedj force << 3 1 for the PMF analysis result of sample 3-a, while rNumber of mass va in the PMF analysis result of sample 3-b I ues matchedj was 1 7. From this, it can be seen that in sample 3, 14 peptide fragments were removed by concentration and separation with Pheny 11 and, as a result, it was possible to detect NBS group-containing peptide fragments. Among the above experimental examples, four types of samples, Sample 1 b, Sample 2-b, Sample 2-c, and Sample 3-b, were tested according to the method of the present invention. For these four types of samples, the EBS protein is the target of analysis, and the NBS reagent that has the ability to modify tributofan residues is used as a modification reagent for isotope labeling, and a phenyl group-containing carrier is used as a carrier for concentration and separation. Analysis. However, the present invention is also applicable to proteins other than E. coli, isotope labeling modifying reagents other than NBS reagents, and phenyl group-containing carriers. The examples of these four samples are therefore merely illustrative in all respects and should not be interpreted in a limited way. Further, all modifications belonging to the equivalent scope of the claims are within the scope of the present invention.

Claims

The scope of the claims

1. (i) preparing a protein sample II and a protein sample II as a control of the protein sample I;

 (ii) preparing two kinds of compounds having different molecular weights by containing isotopes having the same molecular structure and different mass numbers as the modifying reagent, and for the protein sample I, the two kinds The modified protein sample I ′ into which the modifying group derived from any one of the compounds is introduced is modified using either one of the compounds of Modifying one of the two kinds of compounds with the other compound to obtain a modified protein sample II ′ into which a modifying group derived from the other compound is introduced;

 (iii) mixing the modified protein sample I ′ and the modified protein sample I to obtain a mixed sample for electrophoresis;

 (iv) subjecting the mixed sample for electrophoresis to electrophoresis and spreading it into a plurality of protein spots;

(V) A desired protein spot is cut out from the plurality of protein spots, the cut out protein spots are fragmented in the gel, and the peptide fragments containing the modifying group and other peptide fragments are concentrated. (Vi) using a carrier having a structure capable of interacting with a modifying group derived from one of the compounds and a modifying group derived from the other compound; A step of concentrating and separating the modifying group-containing peptide fragment from the mixed sample for concentration and separation to obtain a sample for mass spectrometry containing the concentrated and separated peptide fragment of the modifying group; (vii) Measuring a sample using a mass spectrometer, and identifying and quantifying the concentrated and separated modified fragment-containing peptide fragment; 2007/108140

34 Identification and quantification of protein samples, including

2. In the step (ii), using a compound having an aryl group as a modifying reagent, modified protein I ′ and modified protein II ′ into which the aryl group has been introduced as a modifying group are obtained, The method for identifying and quantifying a protein sample according to claim 1, wherein a carrier having an aryl group is used as a carrier having a structure capable of interacting with the modifying group.

3. The protein sample identification and quantification method according to claim 2, wherein the aryl group of the carrier is a phenyl group.

4. The compound having an aryl group as the modifying reagent is 2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride and 2-nitro [ ¹² C ₆ ] benzenesulfuryl chloride. 3. A method for identifying and quantifying a protein sample according to the above item 2.

5. The protein sample identification and quantification method according to claim 1, wherein, in the step (vi), the carrier is used by being filled or fixed in a pipette tip.

6. In the step (vii), as a matrix,

 3-hydroxymono-4-nitrobenzoic acid and / or —cyanol 3-hydroxycinnamic acid, and

 α-cyan-4-hydroxycinnamic acid,

The protein sample identification and quantification method according to claim 1, wherein a mixture of

7. A tip for concentration and separation, in which a pipette tip is filled or fixed with a carrier having a phenyl group.

8. a tip for concentration separation in which a carrier having a phenyl group is filled or fixed on a pipette tip;

2-nitro [ ¹³ C ₆ ] benzene sulfenyl chloride and 2-nitro [ ¹² C ₆ ] benzene sulfenyl chloride;

Including kit.