JP4165503B2 - Protein quantitative analysis method and kit - Google Patents

Description

  The present invention relates to the field of proteome analysis (overall protein analysis) using stable isotopes.

  In the field of proteome analysis (exhaustive protein analysis), PMF (peptide mass fingerprinting) analysis methods combining two-dimensional electrophoresis and mass spectrometers have been the mainstream. Alternative next-generation proteome analysis methods include, for example, Nature Biotechnology, 994-999, 17, 1999, Molecular & Cellular PROTEOMICS, 299-314, 2, 2003, and Current Opinion in Chemical Biology, 70-77, 7, A technique using stable isotopes as described in 2003 has been devised.

Further, Rapid Communications in Mass Spectrometry, 1642-1650, 17, 2003 and International Publication No. 2004/002950 pamphlet describe a method (NBS method) developed by the present inventors. In NBS method, 2-stable isotopic labels nitrobenzene sulfenyl chloride (NBSCl) (2-nitro ^[13 C _6] benzenesulfenyl chloride) and its non-label (2-nitro ^[12 C _6] benzene sul Phenyl chloride). That is, (1) two types of protein samples, ie, a protein sample I to be analyzed and a control protein sample II thereof, are prepared. (2) The protein sample I is converted into 2-nitro [ ¹³ C ₆ ] benzenesulfenyl. Labeling with either one of chloride and 2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride, and separately separating the protein sample II from 2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride and 2-nitro [ ¹² C ₆ ] labeled with either one of benzenesulfenyl chloride, (3) mixing labeled protein sample I and labeled protein sample II, and (4) obtaining the labeled protein mixture obtained After reduction and alkylation, digest into a peptide mixture containing labeled and unlabeled peptide fragments and (5) label from the peptide mixture The peptide fragments are concentrated and separated using a hydrophobic chromatography column, and (6) mass spectrometry is performed.

An example of the NBS protocol is given below.
・ Two types of protein samples, test sample and control sample, are solubilized in a solution containing 0.1 w / v% SDS and heat-denatured (100 ° C., 3 minutes) ・ NBS (Heavy for one sample ) Add the acetic acid solution in which the reagent is dissolved, add the acetic acid solution in which the NBS (Light) reagent is dissolved to the other sample, and perform the NBS labeling reaction (room temperature, overnight)
・ Mix both samples and remove unreacted reagent using a desalting column (LH-20) ・ Dry the desalted sample and resuspend in a solution containing 0.01 w / v% SDS ・TCEP (Tris (2-carboxyethyl) phosphine hydrochloride) is added to perform the reduction reaction (37 ° C, 30 minutes)
-Add iodoacetamide solution to perform alkylation reaction (room temperature, 45 minutes)
・ Add trypsin and perform site-specific cleavage (37 ℃, 16 hours)
・ Concentrate the NBS-labeled peptide using a concentration column (LH-20) ・ Analyze the concentrated fraction using a mass spectrometer

Steven P. Gygi, Beat List (Beate Rist), Scott A. Gerber, Frantisek Turecek, Michael H. Gelb ), And Ruedi Aebersold, Quantitative analysis of complex protein mixtures using isotope-coded affinity tags, “Nature Biotechnology (Nature Biotechnology) ", 1999, Vol. 17, p. 994-999 Kirk C. Hansen, Gerold Schmitt-Ulms, Robert J. Chalkley, Jan Hirsch, Michael A Baldwin (Michael A. Baldwin) and AL Burlingame, analysis of low-level protein mixtures by mass spectrometry using cleavable 13C isotope-encoded affinity tags and multidimensional chromatography (Mass Spectrometric Analysis of Protein Mixtures at Low Levels Using Cleavable 13C-Isotope-coded Affinity Tag and Multidimensional Chromatography, “Molecular & Cellular PROTEOMICS”, 2003, Vol. 2, p. 299-314 By Salvatore Sechi and Yoshiya Oda, Quantitative proteomics using mass spectrometry, “Current Opinion in Chemical Biology Biology) ", (UK), 2003, Volume 7, p. 70-77 Labeling of tryptophan residues by Hiroki Kuyama, Makoto Watanabe, Chikako Toda, Eiji Ando, Koichi Tanaka, and Osamu Nishimura An Approach to Quantitative Proteome Analysis by Labeling Tryptophan Residues, “Rapid Communications in Mass Spectrometry”, 2003, Vol. 17, p. 1642-1650 International Publication No. 2004/002950 Pamphlet

  The conventional NBS method developed by the present inventors can be used particularly effectively when a purified sample such as a model protein is used. The present inventors can effectively use not only such a purified sample but also a non-purified sample such as a biological sample, and a protein having superior detection sensitivity and quantification than the conventional NBS method. The present invention has been completed to provide a comprehensive quantitative analysis method.

The present invention includes the following inventions.
(1) (i) preparing a protein sample in two states, a protein sample I to be analyzed and a control protein sample II;
(Ii) Solubilized protein sample I is obtained by solubilizing the protein sample I in a solution containing urea as a denaturant or solubilizing in a solution containing guanidine hydrochloride as a denaturant. And
Separately, the step of obtaining the solubilized protein sample II by solubilizing the protein sample II in a solution containing urea as a denaturant or solubilizing in a solution containing guanidine hydrochloride as a denaturant When,
(Iii) the solubilized protein sample I, 2-nitro ^[13 C _6] benzenesulfenyl chloride and 2-nitro ^[12 C _6] be labeled reaction using either benzene sulfenyl chloride To obtain a labeled protein sample I,
Separately, the solubilized protein sample II is subjected to a labeling reaction using one of 2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride and 2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride. Obtaining a labeled protein sample II;
(Iv) obtaining a desalted protein sample mixture by mixing and desalting the labeled protein sample I and labeled protein sample II;
(V) obtaining a resolubilized protein sample mixture by resolubilizing the desalted protein sample mixture with urea or guanidine hydrochloride;
(Vi) obtaining a reduced and alkylated protein sample mixture by reducing and alkylating the resolubilized protein sample mixture;
(Vii) obtaining a peptide mixture containing a labeled peptide fragment and an unlabeled peptide fragment by trypsin digesting the reduced / alkylated protein sample mixture in the presence of urea or guanidine hydrochloride;
(Viii) separating the peptide mixture using a carrier having a phenyl group to obtain a concentrated labeled peptide fragment;
(Ix) A method for comprehensive quantitative analysis of protein, comprising mass spectrometry of the concentrated labeled peptide fragment.

(2) The method according to (1), wherein in the step (ix), mass spectrometry is performed using α-cyano-3-hydroxycinnamic acid or 3-hydroxy-4-nitrobenzoic acid as a matrix.
(3) In the step (ix), when 3-hydroxy-4-nitrobenzoic acid is used as the matrix, the 3-hydroxy-4-nitrobenzoic acid and α-cyano-4-hydroxycinnamic acid are used. The method according to (1), wherein mass spectrometry is performed using the combined mixing matrix.

(4) The method according to (2) or (3), wherein the matrix is used as a solution having a concentration of 1 mg / ml to a saturated concentration.
(5) The method according to (3) or (4), wherein the α-cyano-4-hydroxycinnamic acid is used as a solution having a concentration of 1 mg / ml to a saturated concentration.
(6) The 3-hydroxy-4-nitrobenzoic acid solution and the α-cyano-4-hydroxycinnamic acid solution are used in combination at a volume ratio of 1:10 to 10: 1. ) Method.

(7 ) A kit for performing the method according to any one of (1) to (6) above, wherein the protein sample I to be analyzed and the target protein sample II are labeled. Labeling reagent 2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride, protein I to be analyzed , and target protein sample II labeling reagent 2-nitro [ ¹² C _6] benzenesulfenyl chloride, and obtained by tryptic digestion of the labeled protein sample I and labeled protein sample II, to separate the peptide mixture comprising a labeled peptide fragments and non-labeled peptide fragments A kit comprising a carrier having a phenyl group for obtaining a concentrated labeled peptide fragment .

( 8 ) The kit according to (7 ), further comprising a denaturing agent.
( 9 ) α-cyano-3-hydroxycinnamic acid, 3-hydroxy-4-nitrobenzoic acid, α-cyano-4-hydroxycinnamic acid as a matrix, or 3-hydroxy-4-nitrobenzoic acid and α as a mixed matrix -Kit according to (7) or (8) , further comprising a mixture with cyano-4-hydroxycinnamic acid.
( 10 ) The kit according to ( 8 ) or ( 9 ), wherein the denaturing agent is urea or guanidine hydrochloride.
( 11 ) It further includes at least one selected from the group consisting of a desalting column, a desalting column packed gel, a reducing reagent, an alkylating reagent, trypsin, and a column for packing the carrier. The kit according to any one of ( 10 ).

  According to the present invention, it is possible to provide a method capable of detecting a target peptide from a biological sample with higher sensitivity and higher quantitativeness in mass spectrometry.

  The method of the present invention comprises a sample preparation step (i), a solubilization step (ii), a labeling step (iii), a desalting step (iv), a resolubilization step (v), a reduction / alkylation step (vi ), Digestion step (vii), concentration separation step (viii), and mass spectrometry step (xi).

(I: Sample preparation process)
First, protein samples I and II in two different states are prepared. For example, the protein sample I to be analyzed and the protein sample II can be a sample containing a control protein of the protein contained in the sample I. More specifically, the protein sample I to be analyzed can be a disease state protein sample, and the control protein sample II can be a normal sample protein sample. In the present invention, quantitative analysis of the expression proteome between the protein sample I and the protein sample II is performed. In the present invention, a protein sample to be used as a sample includes a sample having a relatively low molecular weight such as a peptide.

(Ii: solubilization process)
This step is performed separately for protein sample I and protein sample II under the same conditions.

  In this step, a protein sample is solubilized using urea or guanidine hydrochloride which is a denaturing agent. The concentration of the denaturing agent is not particularly limited, and may be appropriately determined by those skilled in the art in consideration of the type of protein sample and other conditions so that the protein sample is solubilized and denatured. For example, urea can be used in an aqueous solution with a concentration of 2M to saturation, preferably 2 to 10M. More preferably, it is used at a concentration of 8M. Guanidine hydrochloride can be used in an aqueous solution of 1.5M to saturated concentration, preferably 1.5 to 8M. More preferably, it is used at a concentration of 6M. Furthermore, the denaturing agent can be used with EDTA of about 5 mM.

  Furthermore, the above-mentioned denaturing agent can be used so as to be 5 to 50 μl per 100 μg of protein sample. A preferable amount is about 25 μl. For example, if the protein sample is in a lyophilized powder state, 25 μl of the denaturing agent may be used, and if the protein sample is in solution, the concentration of the denaturing agent may be adjusted so that the total amount is 25 μl.

  The solubilization temperature is 0 to 30 ° C., preferably about room temperature when urea is used. Moreover, when using guanidine hydrochloride, it is 0-100 degreeC, Preferably it is about room temperature.

(Iii: Labeling process)
In this step, the protein sample solubilized as described above is subjected to two types of labeling reagents, that is, isotope-labeled 2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride (NBSCl (heavy)), Labeling is performed using isotope-unlabeled 2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride (NBSCl (light)). The tryptophan residue in the protein is selectively modified or labeled by the labeling reagent of the present invention. The protein sample I is labeled using either (NBSCl (heavy)) or (NBSCl (light)). Separately, the protein sample II is labeled using either one of (NBSCl (heavy)) and (NBSCl (light)).

  The labeling reagent is preferably used as an acetic acid solution. For example, it can be used as a solution in which 0.17 mg of 2-nitrobenzenesulfenyl chloride is dissolved in 25 μl of acetic acid. Such a labeling reagent can be used in a large excess of about 20 times equivalent to the protein sample. For example, it is good to use so that 2-nitrobenzene sulfenyl chloride in a solution may be 0.17 mg with respect to 100 micrograms of protein samples. As a result, tryptophan residues in the protein sample are labeled almost 100%.

  Strictly speaking, 2-nitrobenzenesulfenyl chloride has a molecular weight difference of about 3% between NBSCl (light) and NBSCl (heavy). However, when the mass of 2-nitrobenzenesulfenyl chloride is described in the present specification, the mass difference between the two in the same substance amount is an amount that can be ignored in calculation. Therefore, both may use the same mass.

The labeling reaction can be performed by adding a labeling reagent to the protein sample and incubating. A reaction time of about 4 hours is sufficient. That is, the reaction may be completed within 4 hours immediately after the start of the reaction. As a standard protocol, the reaction time is preferably 1 hour.
Thus, in the present invention, the labeling reaction time is greatly shortened as compared with the conventional NBS method. For this reason, it is possible to reduce the total work time throughout the protocol from 3 days to 2 days.

(Iv: desalting step)
In this step, the labeled protein sample I and the labeled protein sample II obtained by the above step (iii) are mixed. The resulting protein sample mixture is then desalted. As a desalting method, a conventional method can be used without any particular limitation. For example, a Sephadex LH-20 column may be used and an acetonitrile aqueous solution may be used.

(V: Re-solubilization process)
In this step, the desalted protein sample mixture obtained in the above step (iv) is resolubilized using urea or guanidine hydrochloride which is a denaturing agent. The concentration of the denaturing agent is not particularly limited, and may be appropriately determined by those skilled in the art in consideration of the type of protein sample and other conditions so that the protein sample is solubilized and denatured. For example, urea can be used in an aqueous solution with a concentration of 2M to saturation, preferably 2 to 10M. More preferably, it is used at a concentration of 8M. Guanidine hydrochloride can be used in an aqueous solution of 1.5M to saturated concentration, preferably 1.5 to 8M. More preferably, it is used at a concentration of 6M. In this step, about 50 mM Tris HCl (about pH 8.8) is further added as a buffer and used for pH adjustment in the subsequent reduction / alkylation step.

  In the present invention, by using the conditions as described above in the solubilization step (ii) and the resolubilization step (v), the problem of aggregation that has conventionally occurred in many protein samples can be avoided. Therefore, the solubility can be maintained until the later-described digestion step (vii) with almost no loss of the sample. Examples in which such effects are actually shown are shown in Experimental Examples 1 and 2 described later.

  As described above, according to the method of the present invention, the loss of the sample can be remarkably reduced as compared with the conventional NBS method, so that the NBS-labeled peptide ion detection rate is greatly improved in mass spectrometry. This effect makes it possible to use the present invention usefully when analyzing biological samples such as serum and organ extracts. An example in which such an effect is actually shown is shown in Experimental Example 3 to be described later.

  Furthermore, according to the present invention, the quantitativeness is greatly improved as compared with the conventional NBS method. An example in which such an effect is actually shown is shown in Experimental Example 4 to be described later.

(Vi: Reduction / alkylation process)
In this step, conventional reagents and reaction conditions may be used without any particular limitation. The alkylation reaction mainly occurs on sulfhydryl groups, but can also occur on imidazolyl groups and amino groups. Therefore, in the conventional NBS method, not only an amino acid residue having a sulfhydryl group such as cysteine but also a partial alkylation reaction may occur on an amino acid residue having another amino group. As a result, in the mass spectrum, a peak larger than the peak of the target peptide by m / z = 57 (peak of +57 (m / z)) may be seen. On the other hand, in the present invention, urea or guanidine hydrochloride is contained in the reaction solution in this step. These have an amino group. In the present invention, a partial alkylation reaction that has conventionally occurred for amino acid residues having other imidazolyl groups or amino groups occurs for the amino group of urea or guanidine hydrochloride present in the reaction system. it is conceivable that. That is, it is thought that the alkylation of the side reaction which has occurred in the conventional method is competitively inhibited. As a result, according to the present invention, the +57 (m / z) peak is hardly detected in the mass spectrum. An example in which such an effect is actually shown is shown in Experimental Example 5 described later.

(Vii: digestion process)
In this step, trypsin digestion is performed. This step is performed in the presence of urea or guanidine hydrochloride as a denaturing agent. As the concentration of the denaturing agent, urea can be 0.08 to 4M, preferably 0.8M to 1.6M. Guanidine hydrochloride may be 0.06 to 3M, preferably 0.6M. In this step, it is desirable to add about 5 mM of CaCl ₂ in order to stabilize and activate trypsin structure.

  As the denaturing agent in this step, the residual amount in the sample of the one used in the resolubilization step can be used. For example, a trypsin digestion buffer may be added to the sample after completion of the resolubilization step and the reduction / alkylation step so as to obtain a desired concentration. As another condition for digestion, the pH is adjusted to be close to the optimum pH of the trypsin enzyme. The reaction time may usually be about 4 to 16 hours at 37 ° C. In this way, a peptide mixture containing labeled peptide fragments and unlabeled peptide fragments is obtained.

(Viii: concentration separation process)
In this step, the labeled peptide is selectively concentrated from the peptide mixture by using a carrier having a phenyl group. This step utilizes the inherent selectivity resulting from the π-π electron interaction acting between π-electron compounds. That is, the carrier exhibits an excellent holding ability with respect to the labeled peptide by the interaction between the π electrons of the indole group and nitrophenylthio group of tryptophan in the labeled peptide and the π electrons of the phenyl group in the carrier. , Selective enrichment separation becomes possible. Examples of such carriers include Hi-Trap phenyl FF, Hi-Trap phenyl HP, Phenyl Sepharose 6 Fast Flow, Phenyl Sepharose High Performance (above, manufactured by Amersham Biosciences), YMC * GEL Ph (manufactured by YMC) And can be used as a phenyl column.

  On the other hand, in the conventional NBS method using an LH-20 column, a considerable number of unlabeled peptides were mixed in the eluted fraction. According to the present invention, the number of mixed unlabeled peptides decreases, and in particular, almost no unlabeled peptides in the vicinity of 1200-1700 (m / z), which were found in large numbers in the conventional NBS method. Furthermore, in the conventional NBS method, each peptide is eluted over almost the entire eluted fraction during concentration separation. However, according to the present invention, each peptide can be separated to some extent. As a result, the coverage of the detected peptide is superior to that of the conventional method. An example in which such an effect is actually shown is shown in Experimental Example 6 described later.

(Ix: Mass spectrometry process)
The labeled peptide concentrated and separated by the above step (viii) is subjected to mass spectrometry. When using a MALDI mass spectrometer for the measurement in the present invention, in addition to the MALDI-TOF mass spectrometer (for example, AXIMA-CFR manufactured by Shimadzu Corporation) used in the conventional NBS method, the MAIDI-IT-TOF type A mass spectrometer (for example, AXIMA-QIT manufactured by Shimadzu Corporation) is also used.

  When a MALDI-TOF mass spectrometer is used, 4-CHCA (α-cyano-4-hydroxycinnamic acid) or the like is used as a matrix. On the other hand, when a MAIDI-IT-TOF mass spectrometer is used, the matrix is 3-CHCA (α-cyano-3-hydroxycinnamic acid) or 3H4NBA (3-hydroxy-4- Nitrobenzoic acid (3-hydroxy-4-nitrobenzoic acid) is used.

  These compounds can be appropriately used by those skilled in the art for the purpose of being used as a matrix for mass spectrometry. For example, these compounds are preferably used as a solution. For example, it can be used as a solution of 1 mg / ml to a saturated concentration.

  As a solvent used for preparing such a solution, it is preferable to use an acetonitrile aqueous solution, a trifluoroacetic acid (TFA) aqueous solution, or an acetonitrile-trifluoroacetic acid (TFA) aqueous solution. When an acetonitrile aqueous solution or an acetonitrile-TFA aqueous solution is used, the concentration of acetonitrile is not particularly limited, but 90% or less, preferably about 50% can be used. When a TFA aqueous solution or acetonitrile-TFA aqueous solution is used, the concentration of TFA is not particularly limited, but 1% or less, preferably about 0.1% can be used.

In the case of 4-CHCA, it can be used as a matrix solution of 1 mg / ml to a saturated concentration, preferably 10 mg / ml, by dissolving in such a solvent.
And 3-CHCA can be used as a matrix solution of 1 mg / ml to a saturated concentration, preferably 10 mg / ml, by dissolving in such a solvent.
3H4NBA can be used as a matrix solution having a concentration of 1 mg / ml to a saturated concentration, preferably as a matrix solution having a saturated concentration, by dissolving in such a solvent.

  In the present specification, the standard of the amount expressed in% is v / v% unless otherwise specified.

  In order to identify peptide sequences that differ in abundance between samples, a quadrupole ion trap (QIT) can be used rather than PSD analysis of AXIMA-CFR (manufactured by Shimadzu Corporation) in terms of sensitivity. MS / MS analysis using an on-board mass spectrometer such as AXIMA-QIT (manufactured by Shimadzu Corporation) is preferable. In the measurement of such an ion trap type mass spectrometer, DHB (2,5-dihydroxy benzoic acid) is normally used as a matrix. However, when DHB is used as a matrix, NBS-labeled peptides are hardly detected. Therefore, in the present invention, when performing measurement with an ion trap mass spectrometer, 3-CHCA or 3H4NBA is used as a matrix. This enabled efficient ionization of the NBS-labeled peptide. Therefore, the MS / MS analysis efficiency of the NBS-labeled peptide of the present invention was greatly improved compared to the conventional NBS method. An example in which such an effect is actually shown is shown in Experimental Example 7 to be described later.

  In the present invention, when 3-hydroxy-4-nitrobenzoic acid (3H4NBA) is used as the matrix, 3H4NBA is used as a mixed matrix in combination with α-cyano-4-hydroxycinnamic acid (4-CHCA). preferable. (Hereinafter, in this specification, a matrix that is used alone without using 4-CHCA in combination and a mixed matrix that is used in combination with 4-CHCA may be simply referred to as a matrix.)

The ratio of the combination of 3H4NBA and 4-CHCA is not particularly limited, but can be combined in the following quantitative relationship, for example.
3H4NBA can be prepared in the amounts already mentioned. That is, as a solution having a concentration of 1 mg / ml to a saturated concentration, for example, an acetonitrile aqueous solution, a TFA aqueous solution, or an acetonitrile-TFA aqueous solution is used as a solvent, it can be prepared as a 3H4NBA solution having a concentration of 1 mg / ml to a saturated concentration.
On the other hand, 4-CHCA can also be prepared in the amount already mentioned. That is, when an acetonitrile aqueous solution, TFA aqueous solution, or acetonitrile-TFA aqueous solution is used as a solvent as a solution of 1 mg / ml to a saturated concentration, it should be prepared as a 4-CHCA solution of 1 mg / ml to a saturated concentration, preferably 10 mg / ml. Can do.
Both solutions prepared as described above are preferably used in a volume ratio of 1:10 to 10: 1, more preferably 1: 3 to 3: 1, for example 1: 1.

  The conventional matrix 4-CHCA causes self-disintegration of the measurement sample in measurements with MALDI devices such as MALDI-IT, MALDI-IT-TOF, and MALDI-FTICR devices that require a long time from ionization to ion detection. Despite its drawbacks, it has excellent measurement sensitivity and has the advantage that it is easy to find the optimum spot to which a laser is applied in a mass spectrometry sample.

  On the other hand, the matrix 3H4NBA of the present invention can suppress the progress of self-disintegration of the measurement sample and achieve specific ionization of the hydrophobic sample, particularly the peptide labeled with NBS, as already described. Has the advantage of being able to Then, when the matrix 3H4NBA of the present invention is combined with 4-CHCA, a synergistic effect of the advantages of both matrices is achieved. That is, it is possible to perform mass spectrometry with higher analytical efficiency with the high sensitivity detection capability of 4-CHCA while securing the specific detection capability of 3H4NBA alone. Examples in which such effects are actually shown are shown in Experimental Examples 8 and 9 to be described later.

  The specific protocol of the present invention is shown below. This protocol describes the treatment of 100 μg each of state 1 and state 2 samples. (NBSCl and NBS-labeled peptides should be shielded as much as possible through this protocol.) As mentioned above, unless otherwise specified, the standards for amounts expressed in% in this protocol and the experimental examples shown below are as follows. v / v%.

[Solubilization of samples (separately performed for samples in states 1 and 2)]
1. Prepare 100 μg each of “State 1” and “State 2” samples.
2. Both samples are lyophilized. (Alternatively, dry using a vacuum concentrator.)
3. Both samples are dissolved in 25 μl of denaturation buffer (8M urea aqueous solution containing 5 mM EDTA or 6M guanidine hydrochloride aqueous solution containing 5 mM EDTA).
4). Stir well with a vortex mixer.

[Laboratory labeling of tryptophan residues by NBSCl (performed separately for the state 1 and state 2 samples)]
1. Add 25 μl of NBS reagent (light) solution (acetic acid solution containing 0.17 mg of NBSCl (light)) to the “state 1” sample and stir with a vortex mixer.
Separately, 25 μl of NBS reagent (heavy) solution (acetic acid solution containing 0.17 mg of NBSCl (heavy)) is added to the “state 2” sample and stirred with a vortex mixer.
2. Incubate both samples for 1 hour with gentle agitation.

[Desalination of reaction solution and removal of excess NBS reagent (mixing two kinds of labeled samples in this step)]
1. Prepare an LH-20 column (500 μl of LH-20 equilibrated in advance with 30% acetonitrile in water) and let the supernatant fall down to the height of the resin layer.
2. Gently apply the mixed sample (mixture of sample labeled with NBSCl (light) and sample labeled with NBSCl (heavy), total 100 μl) to the column. (Discard the flow-through fraction.)
3. The column is washed with 100 μl of 30% aqueous acetonitrile.
4). Elute the desalted sample with 200 μl of 30% aqueous acetonitrile.
5. Lyophilize the eluted fraction.

[Reduction / Alkylation]
1. Dissolve the sample in 48 μl of 8 M urea aqueous solution containing 50 mM Tris HCl (pH 8.8) or 6 M guanidine hydrochloride aqueous solution containing 50 mM Tris HCl (pH 8.8).
2. Add 1 μl of reducing solution (200 mM TCEP aqueous solution) and gently stir.
3. Incubate at 37 ° C for 30 minutes.
4). Add 1 μl of alkylating solution (500 mM iodoacetamide in water) and gently stir.
5. Incubate for 45 minutes at room temperature.

[Trypsin digestion]
1. Dissolve 10 μg trypsin (Promega, sequencing grade) in 450 μl of digestion buffer (50 mM Tris HCl (pH 7.8), 5 mM CaCl ₂ ).
2. Add trypsin solution to the sample and pipette to mix gently.
3. Incubate at 37 ° C for 4-16 hours.
4). Add 50 μl (0.1% final concentration) of 1% TFA aqueous solution before loading onto the column. Alternatively, hydrochloric acid may be used instead of TFA. In this case, the final concentration of hydrochloric acid is preferably adjusted to 10 mM.

[Concentration of labeled peptide]
1. Fill an open column with 1 ml of a phenyl column such as Phenyl Sepharose ^™ High Performance (Amersham Biosciences).
2. The column is equilibrated with 5 ml of water followed by 5 ml of 0.1% aqueous TFA.
3. Apply 550 μl of digested sample to the column. (The eluate at this time is collected as a “Flow-through” fraction.)
4). Wash the column with 1 ml of 0.1% TFA aqueous solution. (The eluate at this time is collected as a “Wash” fraction.)
This operation is repeated twice later. (Do three times in total.)
5. Elute with 0.5 ml of elution buffer (0.1% TFA aqueous solution containing 10% acetonitrile) (collect the eluate as “Elute” fraction) and repeat this procedure.
6). Step 5 is repeated until the concentration of acetonitrile is increased by 5% to 40%.
7). (Optional step: Perform as needed.) For MS analysis, desalt and concentrate peptides from the “Flow-through” and “Wash” fractions using ZipTip.
8). The “Elute” fraction is dried by a vacuum concentrator. For further separation using MS analysis or HPLC, the dried sample is suspended in a 0.1% TFA aqueous solution in an amount of about 5 to 50 μl.
In addition, hydrochloric acid may be used in place of the 0.1% TFA aqueous solution used in the concentration of the labeled peptide. In this case, the concentration of hydrochloric acid is preferably adjusted to 10 mM.

[Mass spectrometry]
The sample may be used for MS analysis as it is, or may be further fractionated using HPLC or the like. When performing MALDI-MS measurement, mix the sample with the matrix solution and perform MS measurement.

Furthermore, the present invention provides a kit comprising 2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride, 2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride, and a carrier having a phenyl group. Here, 2-nitro ^[13 C _6] benzene sulfenyl chloride, 2-nitro ^[12 C _6] benzene sulfenyl chloride, Ru used as labeling reagents for carrying out the above SL labeling step (iii). The carrier having a phenyl group, Ru used to implement the above concentration and separation step (viii).

Therefore, the kit of the present invention can be used to carry out comprehensive quantitative analysis of proteins, for example, to carry out the above-described protocol.
That is, the kit of the present invention comprises 2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride (NBSCl (heavy); NBS (heavy) reagent) which is a labeling reagent for carrying out the labeling step (iii) and 2-nitro ^[12 C _6] benzene sulfenyl chloride; wherein the (NBSCl (light) NBS (light ) reagent), the carrier having a phenyl group for carrying out the concentration and separation step (viii), preferably modified Further comprising an agent and / or a matrix. The kit of the present invention may contain various solvents used in the above-described method of the present invention.

  The denaturing agent can be used to carry out the solubilization step (ii) and the resolubilization step (v). Accordingly, as the denaturing agent, urea or guanidine hydrochloride as described in the solubilization step (ii) and the resolubilization step (v) is preferable. The denaturing agent may be dissolved in the solvent described in the solubilization step (ii) and the resolubilization step (v). For example, when the modifying agent is guanidine hydrochloride, it can be provided as dissolved in a solvent. At this time, an aqueous guanidine hydrochloride solution having a concentration of 1.5 M to a saturated concentration, preferably 1.5 to 8 M, more preferably 6 M can be obtained. When urea is dissolved in a solvent as a denaturing agent, an aqueous urea solution having a concentration of 2M to a saturated concentration, preferably 2 to 10M, more preferably 8M can be used.

  The matrix can be used for performing the mass spectrometry step (ix). Therefore, as the matrix, 4-CHCA (α-cyano-4-hydroxycinnamic acid), 3-CHCA (α-cyano-3-hydroxycinnamic acid), and 3H4NBA as described in the mass spectrometric step (ix) are used. It is preferably selected from (3-hydroxy-4-nitrobenzoic acid). Moreover, it is also preferable that it is a set of 3H4NBA and 4-CHCA for use as a mixed matrix.

  These matrices and auxiliary matrices may be dissolved in a solvent as described in the mass spectrometry step (ix). For example, it can be set as a solution of 1 mg / ml to a saturated concentration. When an acetonitrile aqueous solution, TFA aqueous solution or acetonitrile-TFA aqueous solution is used as a solvent, α-cyano-3-hydroxycinnamic acid is 1 mg / ml to saturated concentration, preferably 10 mg / ml, 3-hydroxy-4-nitrobenzoic acid. The acid can be a solution having a concentration of 1 mg / ml to a saturated concentration, preferably a saturated concentration, and α-cyano-4-hydroxycinnamic acid can be a solution having a concentration of 1 mg / ml to a saturated concentration, preferably 10 mg / ml. Among these solutions, a solution of 3-hydroxy-4-nitrobenzoic acid and a solution of α-cyano-4-hydroxycinnamic acid are preferably 1:10 to 10: 1, more preferably 1: A mixed solution mixed in a volume ratio of 3 to 3: 1, for example, 1: 1 may be used.

  Furthermore, the kit of the present invention may further include a desalting column and a desalting column packed gel, a reducing reagent and an alkylating reagent, trypsin, and a column packed with the above-mentioned carrier having a phenyl group. The desalting column and the desalting column-packed gel can be used, for example, to carry out the desalting step (iv) described above. The reducing reagent and the alkylating reagent can be used, for example, for carrying out the above-described reduction / alkylation step (vi). Trypsin can be used, for example, to perform the digestion step (vii). The column packed with the above-mentioned carrier having a phenyl group can be used, for example, for the concentration and separation step (viii). That is, the column packed with the carrier having a phenyl group can be used as a concentration column, and the carrier having the phenyl group can be used as a concentration column-packed gel.

The kit of the present invention enables comprehensive quantitative analysis of the above-described protein of the present invention, and therefore the following effects as already described in the method of the present invention can be obtained.
・ Decrease of sample loss ・ Reduction of +57 (m / z) band ・ Decrease of unlabeled peptide contamination ・ Improvement of separation efficiency of each peptide ・ Realization of MS / MS analysis using QIT ・ Number of detectable pair peaks Increase work time, shorten total work time, and improve quantification

  Experimental examples showing the effects of the present invention using some or all of the steps of the protocol of the present invention described above are shown below.

<Experimental example 1>
Four kinds of purified proteins (ovalbumin ovalbumin (Ova), glyceraldehyde-3-phosphate dehydrogenase (G3P), lysozyme lysozyme (Lys), and α-lactalbumin α-lactalbumin (α- lact), all manufactured by SIGMA), and 25 μg of each were prepared and mixed to give a total of 100 μg as a control sample (C).

  Separately, two types of samples (S) were prepared. One of the samples (S) was prepared as follows. That is, 100 μg of the same protein mixture as the above-mentioned sample (C) was solubilized with 0.1 w / v% SDS aqueous solution containing 5 mM EDTA as in the conventional NBS protocol, and then heated at 100 ° C. for 3 minutes, and NBS (light ) Labeling using a reagent and desalting using an LH-20 column. In addition, the solubilization described in the present experimental example and the following experimental example is a step different from the solubilization generally performed for the sample preparation of SDS-PAGE.

  The other of the samples (S) was prepared as follows. SDS solubilization similar to that described above was performed on 100 μg of the same protein mixture as that of sample (C), and labeling was performed using an NBS (light) reagent. Separately, 100 μg of the same protein mixture as that of the sample (C) was subjected to SDS solubilization as described above, and was labeled using the same amount of NBS (heavy) reagent as the above NBS (light) reagent. Then, the obtained NBS (light) labeled product and NBS (heavy) labeled product were mixed, and desalting was performed using an LH-20 column.

  Separately, two types of samples (G) were prepared. All were obtained in the same manner as in the preparation of the sample (S) described above except that it was solubilized using a 6M guanidine hydrochloride aqueous solution containing 5 mM EDTA as in the protocol of the present invention.

  Separately, two types of samples (U) were prepared. All were obtained by carrying out in the same manner as in the preparation of the sample (S) described above except that it was solubilized using an 8M urea aqueous solution containing 5 mM EDTA as in the protocol of the present invention.

  Electrophoresis was performed on the samples (C), (S), (G), and (U) obtained as described above. For sample (C), an amount corresponding to 10 μg was developed in lane 1. On the other hand, for each of the samples (S), (G) and (U), an amount corresponding to 1/20 was developed (lanes 2 to 7).

  The result of electrophoresis is shown in FIG. In FIG. 1, lane 1 is a control sample (C); lane 2 is a sample containing only NBS (light) labeled protein (S); lane 3 is a sample containing only NBS (light) labeled protein (G); lane 4 is a sample (U) containing only NBS (light) labeled protein; lane 5 is a sample (S) in which NBS (light) labeled protein and NBS (heavy) labeled protein are mixed in equal amounts; lane 6 is NBS (light) Sample in which equal amounts of labeled protein and NBS (heavy) labeled protein are mixed (G); Lane 7 is a sample in which equal amounts of NBS (light) labeled protein and NBS (heavy) labeled protein are mixed It is about (U). As shown in the results of lanes 3, 4, 6 and 7 in FIG. 1, when guanidine hydrochloride or urea, which is a denaturing agent, is used for solubilization, the sample can be kept soluble with almost no loss. Indicated.

<Experimental example 2>
100 μg of mouse (C57BL) serum was used as a control sample (C).

  Separately, two types of samples (S) were prepared. One of the samples (S) was prepared as follows. That is, 100 μg of the same mouse serum as the above sample (C) was solubilized with a 0.1 w / v% SDS aqueous solution containing 0.5 mM EDTA as in the conventional NBS protocol, and then heated at 100 ° C. for 3 minutes, and NBS ( light) Labeling was performed using a reagent. Separately, 100 μg of the same mouse serum as sample (C) was subjected to SDS solubilization in the same manner as described above, and labeled with the same amount of NBS (heavy) reagent as the above NBS (light) reagent. And the obtained NBS (light) labeled product and the NBS (heavy) labeled product were mixed to obtain a labeled mixture.

  The other of the samples (S) was prepared as follows. That is, a labeled mixture was prepared in the same manner as described above for 100 μg of mouse serum, and further obtained by desalting with an LH-20 column.

  Separately, two types of samples (G) were prepared. All were obtained in the same manner as in the preparation of the sample (S) described above except that it was solubilized using a 6M guanidine hydrochloride aqueous solution containing 5 mM EDTA as in the protocol of the present invention.

  Separately, two types of samples (U) were prepared. All were obtained by carrying out in the same manner as in the preparation of the sample (S) described above except that it was solubilized using an 8M urea aqueous solution containing 5 mM EDTA as in the protocol of the present invention.

  The obtained samples (C), (S), (U) and (G) were subjected to electrophoresis. As for sample (C), an amount corresponding to 10 μg was developed in lane 2 and an amount corresponding to 2 μg was developed in lane 3. Samples (S), (U) and (G) were all developed in an amount corresponding to 1/20 (lanes 4 to 9).

  The result of electrophoresis is shown in FIG. In FIG. 2, lane 1 is a molecular weight marker, lanes 2 and 3 are control samples (C); lane 4 is a sample after labeling (S); lane 5 is a sample after labeling and desalting (S); lane 6 Is labeled sample (U); lane 7 is labeled and desalted sample (U); lane 8 is labeled sample (G); lane 9 is labeled and desalted sample (G) ). As shown in the results of lanes 6, 7, 8, and 9 in FIG. 2, when guanidine hydrochloride or urea as a denaturing agent is used for solubilization, the sample can be kept soluble with almost no loss. Indicated.

<Experimental example 3>
Mouse liver extract was used as an analysis sample. According to the conventional NBS protocol, this analysis sample was solubilized, labeled, desalted, resolubilized with SDS, reduced / alkylated, and digested. Separately, according to the protocol of the present invention, the analysis sample was solubilized, labeled, desalted, resolubilized with urea, reduced / alkylated, and digested. In any labeling operation, on the one hand, the solubilized sample is labeled with an NBS (light) reagent; on the other hand, an equal amount of the solubilized sample is equivalent to the NBS (light) reagent in the same amount. (Heavy) Reagent was used for labeling; then, both of the obtained labeled products were mixed. Also in the following experimental examples, the same labeling operation is performed. NBS-labeled peptides were separated from each of the obtained samples using phenyl column (Amersham Biosciences column; HiTrap phenyl). Elution was performed with a step concentration gradient of acetonitrile (specifically, it was carried out for 10 steps of 40% and 7 steps of concentration every 5%), and two fractions were fractionated at each concentration.

  Each elution fraction (EL1 to EL14) was analyzed using AXIMA-CFR, and the number of observed pair peaks was summarized in Table 1. In the table, (a) shows the results for the method using SDS, and (b) shows the results for the method using urea.

  As this table shows, only 14 pair peaks were observed in the method using SDS as in the conventional NBS protocol, but 76 pair peaks in the method using urea as in the protocol of the present invention. Observed. Moreover, the mass spectrum about EL5 fraction (namely, acetonitrile concentration 20%) in a table | surface is shown to Fig.3 (a) and (b). In the figure, the peak indicated by the arrow is a paired peak of the labeled peptide.

<Experimental example 4>
A mixture of three types of purified proteins (G3P, Lys and α-lact) was used as a model protein. This mixture was solubilized with SDS, labeled, desalted, re-solubilized with SDS, reduced / alkylated and digested according to the conventional NBS protocol. Separately, the mixture was solubilized with guanidine hydrochloride, labeled, desalted, resolubilized with guanidine hydrochloride, reduced / alkylated, and digested according to the protocol of the present invention. Furthermore, the same operation as that according to the above-described protocol of the present invention was performed on the mixture, except that urea was used for solubilization. For each of the obtained samples, NBS-labeled peptides were separated using phenyl column, and MS analysis was performed.

  About the obtained MS spectrum, the area ratio of the monoisotopic peak (monoisotopic peak) of each pair peak was quantified and compared. Specifically, of the paired peaks, the relative area of the smaller peak when the area of the larger peak is defined as 100 was obtained, and both spectra were compared using this as an index of quantitativeness. The results are shown in Table 2. As shown in Table 2, the average value of the relative area of the peak is 80.3 in the method using SDS as in the conventional NBS protocol, whereas guanidine hydrochloride (GdnHCl) and urea are as in the protocol of the present invention. In the method using (Urea), they were 90.0 and 92.6, respectively. From this, it was shown that the quantitative property was greatly improved by the present invention.

  Furthermore, the area ratio dispersion and standard deviation of the pair peak were obtained. The results are also shown in Table 2. As shown in Table 2, the dispersion is 186.4 in the method using SDS as in the conventional protocol, whereas it is 37.1 in the method using guanidine hydrochloride and urea as in the protocol of the present invention, respectively. 30.5. On the other hand, the standard deviation was 13.7 in the method using SDS as in the conventional protocol, whereas it was 6.1 and 5.5 in the method using guanidine hydrochloride and urea as in the protocol of the present invention, respectively. . The smaller the variance and the standard deviation, the smaller the data variation. Therefore, it was shown that such a variation was greatly improved by the method of the present invention.

<Experimental example 5>
Using a mixture of four types of purified proteins (Ova, G3P, Lys, and α-lact) as model proteins, solubilization, labeling, desalting, resolubilization, reduction / alkylation and digestion are performed according to the conventional NBS protocol. went. On the other hand, using the same model protein, solubilization with urea, labeling, desalting, resolubilization with urea, reduction / alkylation and digestion were performed according to the protocol of the present invention. Furthermore, each sample was fractionated using phenyl column, and one fraction was collected from those prepared by operating according to the conventional NBS protocol, and prepared according to the protocol of the present invention. For the sample, a fraction corresponding to the one fraction was collected. Each collected fraction was analyzed using AXIMA-CFR. The results obtained at this time are shown in FIGS. 4 (a) and 4 (b). In FIG. 4, (a) shows the result by the conventional NBS protocol, (b) shows the result by the protocol of the present invention, and each figure shows the mass / charge (Mass / Charge) on the horizontal axis and the ion on the vertical axis. Represents relative intensity. As shown in the results of FIG. 4, in the conventional method, a +57 (m / z) peak indicating that a side reaction alkylation occurred was detected, but in the method of the present invention, such a peak is Not detected.

<Experimental example 6>
Using a mixture of 4 types of purified proteins (Ova, G3P, Lys and α-lact) as model proteins, solubilization, labeling, desalting, resolubilization, reduction / alkylation, digestion, and concentration according to conventional NBS protocols The operation of separating NBS-labeled peptides using a column (LH-20) was performed. A mass spectrum of a typical elution fraction is shown in FIG. On the other hand, using the same model protein, solubilization with urea, labeling, desalting, resolubilization with urea, reduction / alkylation, digestion, and phenyl column (Amersham Biosciences column; HiTrap phenyl) The operation of NBS-labeled protein separation using was performed. A mass spectrum of a typical elution fraction is shown in FIG.

  In each figure, the horizontal axis represents mass / charge (Mass / Charge), and the vertical axis represents the relative intensity of ions. Fig. 5 shows the results for the 1st, 3rd, 5th, 7th and 9th fractions (Fr.1, Fr.3, Fr.5, Fr.7 and Fr.9) of all 10 fractions by LH-20. It is. FIG. 6 shows the first, fourth, seventh, tenth, twelfth and fourteenth fractions (Fr.1, Fr.4, Fr.7, Fr.10, Fr.12 and Fr. It is a result about 14). As shown in the results of FIGS. 5 and 6, in the method of the present invention, almost no unlabeled peptide around 1200-1700 (m / z), which was frequently observed in the conventional NBS method, was detected. In FIG. 5, the first, second, fourth, ninth, tenth, twelfth and thirteenth peptides (indicated by arrows in the figure) were eluted over almost the entire eluted fraction. In 6, the peptides were separated to some extent and eluted.

<Experimental example 7>
Using a mixture of 4 types of purified proteins (Ova, G3P, Lys and α-lact) as model proteins, solubilization with urea, labeling, desalting, resolubilization with urea, reduction and alkylation according to the protocol of the present invention, Digestion and NBS-labeled peptide separation using phenyl column were performed. Next, one fraction eluted with phenyl column was prepared as a sample for mass spectrometry, and mass spectrometry by AXIMA-QIT was performed for each of the following three types of matrices. As the matrix, three types of DHB used conventionally, 3-CHCA and 3H4NBA used in the present invention were dissolved in 50% acetonitrile aqueous solution containing 0.1% TFA, respectively. ml, 3H4NBA was used as a saturated solution. An equal amount of the prepared sample and the matrix solution were mixed and measured with AXIMA-QIT. The results obtained at this time are shown in FIGS.

  In each figure, the horizontal axis represents mass / charge (Mass / Charge), the vertical axis represents the relative intensity of ions, and the peaks indicated by arrows (i) to (iii) in FIGS. , A paired peak of labeled peptides. As shown in FIGS. 7A to 7C, when DHB is used as a matrix (a), NBS-labeled peptide is hardly detected because it is not ionized, but 3-CHCA (b) When 3H4NBA (c) was used, the NBS-labeled protein was efficiently ionized, so that detection on the mass spectrum became possible. Further, the results of MS / MS analysis of the ions corresponding to 1118.53 (m / z) corresponding to the NBS (light) labeled peptide among the paired peaks indicated by the arrow (i) obtained in FIG. It is shown in FIG.

<Experimental Example 8>
In this experimental example, a mixture of a peptide modified with an NBS reagent and an unmodified peptide was used as a measurement sample, and measurement was performed with a mass spectrometer using a mixed matrix of 3H4NBA and 4-CHCA.

The measurement sample was prepared as follows.
Two types of purified proteins (ovalbumin, glyceraldehyde-3-phosphate dehydrogenase, lysozyme, α-lactalbumin; all manufactured by SIGMA) were mixed at 25 μg each to give a total of 100 μg. Using urea final concentration 8M as modifiers, except for performing the re-solubilization of solubilization and NBS modified sample mixture for each of the mixtures, measured according to ^"13 CNBS Isotope Labeling Kit" (manufactured by Shimadzu Corporation) protocol Samples were prepared. That is, one is labeled with NBS Reagent (heavy) (2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride) and the other is labeled with NBS Reagent (light) (2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride). Unlabeled modification, mixing of both modified samples, desalting, resolubilization with urea, reduction, alkylation, and trypsin digestion were performed. The sample after digestion was desalted with ZipTipμ-C18 and eluted with 4 μl of 50% acetonitrile aqueous solution containing 0.1% TFA as a measurement sample. Of this, 0.5 μl was applied on a mass plate.

  A matrix prepared as follows was used. 3H4BA and 4-CHCA were dissolved in 50% acetonitrile aqueous solution containing 0.1% TFA as a solvent. 3H4NBA was a saturated solution, and 4-CHCA was a 10 mg / ml solution. The solution thus prepared was mixed at a volume ratio of 1: 1 to obtain a matrix mixed solution. MALDI-IT-TOF mass spectrometer with ion trap (AXIMA-QIT, Shimadzu Corporation) after adding 0.5 μl of the matrix mixed solution to the mass plate with the measurement sample applied in advance and drying And a MALDI-TOF mass spectrometer (AXIMA-CFR plus, manufactured by Shimadzu Corporation) without an ion trap.

The MS spectrum obtained at this time is shown in FIG. In FIG. 9, the horizontal axis represents mass / charge (m / z), and the vertical axis represents the relative intensity (% Int.) Of ions. Further, (a) is a spectrum obtained by measurement with AXIMA-QIT having an ion trap, and (b) is a spectrum obtained by measurement with AXIMA-CFR plus without an ion trap. In FIG. 9, the peak pair marked with an arrow indicates that the peptide is an NBS-modified peptide. Each paired peak is composed of two modifying reagents NBS Reagent (heavy) (2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride) and NBS Reagent (light) (2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride). It has a difference of m / z = 6 corresponding to the mass difference.

  As can be seen by comparing the spectra of (a) and (b) of FIG. 9 with each other, almost the same spectra were obtained in both. The fact that almost the same spectrum can be obtained by a mass spectrometer with an ion trap and a mass spectrometer without an ion trap means that the self-disintegration of the measurement sample is suppressed in the measurement of the mass spectrometer with an ion trap. Indicates. That is, when the matrix mixture of the present invention is used, in the conventional measurement with an ion trap type mass spectrometer (ie, a mass spectrometer having a relatively long time from ionization to ion detection) using 4-CHCA alone as a matrix. It was found that the self-disintegration of the measurement sample that had occurred was suppressed. In addition, since most of the detected peaks were NBS-modified peptide pair peaks, it was found that the specific detectability of matrix 3H4NBA alone was retained even when a mixed matrix was used. .

<Experimental Example 9>
In this experimental example, the same measurement sample as in experimental example 8 was used, and measurement was performed with a mass spectrometer using a matrix using 3H4NBA alone and a mixed matrix of 3H4NBA and 4-CHCA.

  Using the same sample as in Experimental Example 8, 4 μl of eluate was obtained as a measurement sample by the same operation. Of this, 1 μl was diluted 1000-fold with a 0.1% TFA aqueous solution, and 0.5 μl of that was applied onto a mass plate.

As the matrix, a matrix using 3H4NBA alone and a mixed matrix combining 3H4NBA and 4-CHCA were used.
For the matrix using 3H4NBA alone, 3H4NBA was dissolved in 50% acetonitrile aqueous solution containing 0.1% TFA to obtain a saturated solution. This 3H4NBA solution is added to a mass plate with a measurement sample applied in advance, dried, and then measured with a MALDI-TOF mass spectrometer (AXIMA-CFR plus, manufactured by Shimadzu Corporation). It was.
A mixed matrix of 3H4NBA and 4-CHCA was prepared in the same manner as in Experimental Example 8. This mixed solution is added to a mass plate with a measurement sample applied in advance, dried, and then measured with a MALDI-TOF mass spectrometer (AXIMA-CFR plus, manufactured by Shimadzu Corporation). It was.

FIG. 10 shows the MS spectrum obtained at this time. In FIG. 10, the horizontal axis represents mass / charge (m / z), and the vertical axis represents the relative intensity (% Int.) Of ions. In addition, (a) is a spectrum obtained by using 3H4NBA and 4-CHCA as a matrix and (b) is a spectrum obtained by using 3H4NBA as a matrix. Further, the peak pair marked with an arrow in FIG. 10 indicates that the peptide is an NBS-modified peptide. Each paired peak consists of two labeling reagents, NBS Reagent (heavy) (2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride) and NBS Reagent l (ight) (2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride). M / z = 6, which corresponds to the mass difference between

  As can be seen by comparing the spectra of (a) and (b) in FIG. 10, a pair peak of the NBS-modified peptide is detected in (a) with higher sensitivity than in (b). This indicates that detection can be performed with higher sensitivity by further mixing 4-CHCA with 3H4NBA as a matrix. That is, while 3H4NBA has the feature that “specific detection of NBS-modified peptide can be performed in mass spectrometry”, 4-CHCA can be easily searched for the optimum spot to which a laser is applied. We were able to confirm that the feature that “highly sensitive measurement can be performed” was added.

  Combining the results of Experimental Example 8 and Experimental Example 9 described above, the characteristics of 3H4NBA are “specific detection of NBS-modified peptide in mass spectrometry” and “relative time from ionization to ion detection. It is possible to suppress the self-disintegration of the sample even if it is measured with a long ion trap type MALDI mass spectrometer ”, and the state that“ the best spot to shine the laser can be easily found ”is a feature of 4-CHCA Thus, it was confirmed that “a measurement with good sensitivity” was achieved at the same time.

2 is an electrophoretogram obtained in Experimental Example 1. FIG. 6 is an electrophoretogram obtained in Experimental Example 2. FIG. 4 is an MS spectrum obtained in Experimental Example 3. 6 is an MS spectrum obtained in Experimental Example 5. 7 is an MS spectrum obtained in Experimental Example 6. 7 is an MS spectrum obtained in Experimental Example 6. 7 is an MS spectrum obtained in Experimental Example 7. 10 is an MS / MS spectrum obtained in Experimental Example 7. 10 is an MS spectrum obtained in Experimental Example 8. 10 is an MS spectrum obtained in Experimental Example 9.

Claims (11)

(I) preparing a protein sample in two states, a protein sample I to be analyzed and a control protein sample II thereof;
(Ii) Solubilized protein sample I is obtained by solubilizing the protein sample I in a solution containing urea as a denaturant or solubilizing in a solution containing guanidine hydrochloride as a denaturant. And
Separately, the step of obtaining the solubilized protein sample II by solubilizing the protein sample II in a solution containing urea as a denaturant or solubilizing in a solution containing guanidine hydrochloride as a denaturant When,
(Iii) the solubilized protein sample I, 2-nitro ^[13 C _6] benzenesulfenyl chloride and 2-nitro ^[12 C _6] be labeled reaction using either benzene sulfenyl chloride To obtain a labeled protein sample I,
Separately, the solubilized protein sample II is subjected to a labeling reaction using one of 2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride and 2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride. Obtaining a labeled protein sample II;
(Iv) obtaining a desalted protein sample mixture by mixing and desalting the labeled protein sample I and labeled protein sample II;
(V) obtaining a resolubilized protein sample mixture by resolubilizing the desalted protein sample mixture with urea or guanidine hydrochloride;
(Vi) obtaining a reduced and alkylated protein sample mixture by reducing and alkylating the resolubilized protein sample mixture;
(Vii) obtaining a peptide mixture comprising a labeled peptide fragment and an unlabeled peptide fragment by trypsin digesting the reduced / alkylated protein sample mixture in the presence of urea or guanidine hydrochloride;
(Viii) separating the peptide mixture using a carrier having a phenyl group to obtain a concentrated labeled peptide fragment;
(Ix) A method for comprehensive quantitative analysis of protein, comprising mass spectrometry of the concentrated labeled peptide fragment.   The method according to claim 1, wherein in the step (ix), mass spectrometry is performed using α-cyano-3-hydroxycinnamic acid or 3-hydroxy-4-nitrobenzoic acid as a matrix.   In the step (ix), when 3-hydroxy-4-nitrobenzoic acid is used as the matrix, mixing of the 3-hydroxy-4-nitrobenzoic acid and α-cyano-4-hydroxycinnamic acid The method according to claim 1, wherein mass spectrometry is performed using a matrix. The matrix is used as a solution having a concentration of 1 mg / ml to a saturated concentration.
The method described in 1.   The method according to claim 3 or 4, wherein the α-cyano-4-hydroxycinnamic acid is used as a solution having a concentration of 1 mg / ml to a saturated concentration.   The solution of the 3-hydroxy-4-nitrobenzoic acid and the solution of the α-cyano-4-hydroxycinnamic acid are used in combination at a volume ratio of 1:10 to 10: 1. the method of. A kit for performing the method according to any one of claims 1 to 6,
2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride, which is a labeling reagent for labeling either the protein sample I to be analyzed or the target protein sample II ,
2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride, which is a labeling reagent for labeling either the protein I to be analyzed or the target protein sample II , and
Labeled peptide fragments enriched by separating a peptide mixture comprising labeled peptide fragments and unlabeled peptide fragments obtained by trypsin digestion of labeled protein sample I and labeled protein sample II A kit comprising a carrier having a phenyl group to obtain The kit according to claim 7, further comprising a denaturing agent. Α-cyano-3-hydroxycinnamic acid, 3-hydroxy-4-nitrobenzoic acid, α-cyano-4-hydroxycinnamic acid as matrix, or 3-hydroxy-4-nitrobenzoic acid and α-cyano- as mixed matrix The kit according to claim 7 or 8 , further comprising a mixture with 4-hydroxycinnamic acid. The kit according to claim 8 or 9 , wherein the denaturing agent is urea or guanidine hydrochloride. The desalting column, the desalting column packed gel, the reducing reagent, the alkylating reagent, trypsin, and at least one selected from the group consisting of columns for packing the carrier, further comprising any one of claims 7 to 10 The kit according to claim 1.