WO2008015768A1

WO2008015768A1 - Methods for quantifying oxidized tryptophan residues in sample and for determining the location thereof

Info

Publication number: WO2008015768A1
Application number: PCT/JP2006/315693
Authority: WO
Inventors: Hiroki Kuyama; Osamu Nishimura
Original assignee: Shimadzu Corporation
Priority date: 2006-08-02
Filing date: 2006-08-02
Publication date: 2008-02-07
Also published as: JPWO2008015768A1; JP4656236B2

Abstract

It is intended to provide a method whereby the degree of oxidization of tryptophan residues in a protein or a peptide can be determined and a method whereby the location of oxidized tryptophan residues can be determined. Namely, a method of quantifying oxidized tryptophan residues in proteins which comprises: (i) preparing a protein sample A containing tryptophan residues the degree of oxidization of which is unknown and another protein sample B containing tryptophan residues the degree of oxidization of which is unknown; (ii) modifying the tryptophan residues in the protein sample A by using one of a heavy reagent and a light reagent to be used in the isotope labeling method to give a modified protein sample A’, while separately modifying the tryptophan residues in the protein sample B by using the other of the heavy reagent and the light reagent as described above to give a modified protein sample B’; (iii) mixing the modified protein sample A’ with the modified protein sample B’ to give a sample mixture for mass spectroscopy; and (iv) subjecting this sample mixture for mass spectroscopy to mass spectroscopic analysis to thereby determine the degree of oxidization of tryptophan residues in each of the protein sample A and the protein sample B.

Description

Description Method for quantifying and locating oxidized tributophan residues in proteins Technical Field

The present invention relates to the field of protein'peptide chemistry, and the field of mass spectrometry. More specifically, the present invention relates to a method for quantification and positioning of oxidized tributophane residues in a protein (Method for Quantification and Positioning of Oxidized Tryptophan Residue in Protein). Background art

Conventionally, quantification of oxidized tributophane residues has been performed by measuring UV-visible spectra and analyzing changes in absorbance due to oxidation (for example, WE Savi e and A. Fontana, International Journal of Peptide and Protein Research, 15, 285 (1980)).

— On the other hand, sulfenyl halides (for example, Scoffone E, Fontana A, Rocchi R, Sulfenyl Halides as Modifying Reagents for Polypeptides and Proteins. Modification of Tryptophan Residues, Biochemistry, 7 (1968) 971-979) and benzyl halides (eg Horton, HR and Koshland, D. E, Jr. (1972), Modification of proteins with acti e benzyl hal ides, Methods in ENZYM0L0GY, 25, 468-482) are known.

Quantitative proteomic analysis focusing on tributophan residues in protein 'peptides has been performed using sulfenyl halide stable isotope-labeled compounds (for example, Rapid Communications in Mass Spectrometry, 17, 1642-1650). (2003), International Publication No. 2004/002950 pamphlet). Non-patent literature 1 WE Savige and A. Fontana, “International Journal of Peptide and Protein Research” 1980, Volume 15, p.285

Non-Patent Document 2 Scophone ■ E (Scoff one E), Fontana 'A (Fontana A), and Rocchi' R (Rocchi R), "Biochemi stry j, Biochemi stry j, ί968, Vol. 7, p. 971-979

Non-Patent Document 3 Houghton ■ H ■ R (Horton, H.R.) ¾ and Koshland ■ D

E. Junior (Koshland, D. E, J), Γ Methods. In Enzymology (Methods in ENZYM0L0GY) j, 1972, Vol. 25, pp.468-482

Non-Patent Document 4 Γ Rapid 'Communications in Mass Spectrometry' 2003, Vol. 17, p.1642-1650

Patent Document 1 International Publication No. 2004/002950 Pamphlet 開示 Disclosure of Invention

Object of the invention

It has been reported that the oxidation of proteins occurring in vivo causes a decrease in protein activity and the development of toxicity, and is associated with cataracts, emphysema, rheumatism, asthma and the like. Furthermore, protein oxidation does not proceed uniformly at any position, but there are positions that are susceptible to oxidation and positions that are not. Knowing the position and extent of oxidation under various ίη vivo or in vitro conditions is considered to have great significance in elucidating life phenomena and structure-activity relationships. In the present invention, attention was paid to the degree of oxidation of the tribtophan residue. Tryptophan residue is an amino acid residue that is easily oxidized along with methionine residue, and has attracted attention in studies such as oxidative stress and age-related changes. Among the conventional techniques mentioned above, the method for quantifying tributophane oxide residues using the UV-visible spectrum cannot perform accurate quantification. It is also unclear which tributophane residues in the protein are oxidized.

Of the conventional technologies listed above, quantitative proteome analysis using a stable isotope-labeled compound of sulfinyl halide does not suggest quantification of oxidized tributophane residues. An object of the present invention is to provide a method capable of determining the degree of oxidation of a tributophane residue in a protein peptide and a method capable of determining the position of the oxidized tributophane residue. is there.

Summary of the Invention

The present invention includes the following inventions.

The following inventions (1) to (4) are methods for quantifying the relative amount of protein oxidation.

(1) (i) Prepare a protein sample A in which the degree of oxidation of tribtophan residues is unknown and a protein sample B in which the degree of oxidation of tribtophan residues is unknown,

(ii) As modifying agents, two types of compounds having the same molecular structure and having different molecular weights by containing isotopes having different mass numbers are prepared. Tributofan residue is modified using one of the compounds of the species to obtain a modified protein sample A ′, and separately from the protein sample B, the other of the two compounds The tributofane residue was modified with the compound of, and the modified protein sample B ′ was obtained.

(iii) Mixing the modified protein sample A ′ and the modified protein sample B ′ to obtain a mixed sample for mass spectrometry,

(IV) subjecting the mixed sample for mass spectrometry to mass spectrometry and quantifying the degree of oxidation of tryptophan residues in each of protein sample A and protein sample B, and oxidizing tributophan residues in the protein To perform quantitative determination. In the above (1), the term “protein” is used to mean a peptide including 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 modified separately using two types of modifying reagents that differ only in the isotopic composition in step (U). (In other words, one protein sample isotope-labeled by the isotope labeling method), two kinds of modified protein samples are mixed in step (iii), and mass spectrometry is performed in step (iv).

In the above (1), the mixed sample for mass spectrometry obtained in the step (iii) may be appropriately processed according to the type of the sample to be subjected to the method of the present invention. That is, in the step (Mi), a process selected from a desalting process, a drying process, a re-solubilization process, a reductive alkylation process, a fragmentation process, a concentration separation process, a fractionation process, and the like, together with the mixing process by a person skilled in the art. It may be performed appropriately.

(2) The method for quantifying an oxidized tributophan residue in a protein according to (1), wherein the modifying reagent is a sulfenyl compound. (3) The sulfenyl compound is arylsulfenyl halide. (2) A method for quantifying oxidized tributophan residues in the protein according to 1.

(4) The method for quantifying an oxidized tributophan residue in a protein according to (3), wherein the arylsulfuryl halide is 2-nitrobenzenesulfuryl chloride. The invention (5) below is a method for determining the oxidation position of a tributophane residue in a protein.

(5) A method for determining the position of an oxidized tributophan residue by performing the method according to any one of (1) to (4) and then performing amino acid sequencing.

The inventions (6) to (9) below are methods for quantifying the absolute amount of protein oxidation.

(6) (i) Prepare a protein sample C for which the degree of oxidation of tribtophan residues should be quantified and a protein sample D for which the degree of oxidation of tribtophan residues is known,

(i i) As a modifying reagent, two types of compounds having the same molecular structure and different molecular weights by containing isotopes having different mass numbers are prepared.

The protein sample C is modified with a tributophane residue using either one of the two kinds of compounds to obtain a modified protein sample C ′. Tributofan residue is modified with the other of the two compounds to obtain a modified protein sample D ′,

(iii) Mixing the modified protein sample C ′ and the modified protein sample D ′ to obtain a mixed sample for mass spectrometry, (iv) Quantifying the oxidized tributophane residue in the protein, comprising quantifying the degree of oxidation of the tributophane residue in the protein sample C by subjecting the mixed sample for mass spectrometry to mass spectrometry How to do. (6) I. In this context, protein is used in the sense that it includes peptides with relatively low molecular weight.

In (6) above, two types of protein samples are prepared in step (i), and the two types of protein samples are prepared separately using two types of modifying reagents that differ only in the isotopic composition in step (ii). Decorate (ie, label one of the protein samples by isotope labeling), mix the two modified protein samples in step (iii), and perform mass spectrometry in step (iv).

In the above (6), the mixed sample for mass spectrometry obtained in the step (i i i) may be appropriately processed according to the type of the sample used in the method of the present invention. That is, in the step (iii), a process selected from a desalting process, a drying process, a re-solubilization process, a reductive alkylation process, a fragmentation process, a concentration separation process, a fractionation process, and the like together with a mixing process by a person skilled in the art. It may be performed appropriately.

(7) The method for quantifying an oxidized tributophan residue in a protein according to (6), wherein the modifying reagent is a sulfenyl compound.

(8) The method for quantifying an oxidized tributophan residue in a protein according to (7), wherein the sulfenyl compound is aryl sulfenyl halide.

(9) Oxidized rib ribophane residue in the protein according to (8), wherein the arylsulfuryl halide is 2-nitrobenzenesulfuryl chloride To perform quantitative determination. The following invention (10) is a method for determining the oxidation position of a tributophan residue in a protein.

(10) A method for determining the position of an oxidized tributophan residue by performing the method according to any one of 6) to (9) and then determining the amino acid sequence.

Brief Description of Drawings

FIG. 1 is a diagram outlining an example of the case where protein sample A and protein sample B, in which the oxidation of tribtophan residues are both unknown, are analyzed in the present invention.

FIG. 2 shows that in the present invention, protein sample C in which the degree of oxidation of the tribtophan residue is unknown and that the tritophane residue is not oxidized (that is, the degree of oxidation is 0 0/0). FIG. 4 is a diagram outlining an example when a non-oxidized protein sample D is an analysis target.

Figure 3 shows an MS spectrum showing the results of quantification of the degree of tributofan residue oxidation according to the present invention using a DSIP sample in which tributofan residue non-oxidized DSIP sample was oxidized with 50% of tributophane residue. It is Kutor.

BEST MODE FOR CARRYING OUT THE INVENTION

(i: Preparation of protein sample)

First, two types of protein samples are prepared for examining the oxidation state of tribtophan residues. The two types of protein samples should include the following relationships: You can.

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 one of the above proteins: 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 degree of oxidation of at least one of the two proteins is unknown, and the average molecular weight of the proteins in both protein samples will differ strictly if the degrees of oxidation of the two are different. However, since the tributophane residue targeted in the present invention is an amino acid belonging to a class with the lowest occurrence frequency in proteins, the difference in the average molecular weight is considered to be essentially negligible. be able to.

When the degree of oxidation of tributophan residues is unknown in both of the two protein samples (in this case, the two protein samples may be referred to as protein samples A and B), It is possible to know the relative degree of oxidation of a protein sample. When the oxidation degree of one of the two protein samples is unknown and the oxidation degree of the other is known (in this case, one protein sample is protein sample C and the other protein sample is protein sample D) It is possible to know the absolute degree of oxidation of the one protein sample. The protein sample may have been solubilized. Solubilization method There is no particular limitation. 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 the denaturing agent is not particularly limited, and may be appropriately determined by a person skilled in the art in consideration of the type of the protein sample and other conditions so that the protein sample is solubilized and denatured. 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, a non-oxidized tributophan residue is modified in both types of proteins by a modifying reagent for isotope labeling.

As the modifying reagent, a compound that has the ability to selectively modify tributophane residues and does not react with oxidized tributophane residues can be used without particular limitation.

Tribtophan residues are most reactive at position 2 of the side chain indole ring, and oxidation usually occurs at this position. Therefore, as a modifying reagent for the tributophane residue used in the present invention, when the tributofan residue is not oxidized, it has reactivity to the 2-position of the side chain indole ring, and the tributophane residue is oxidized. A compound having such a non-reactive characteristic is used.

In addition, the modifying reagent in the present invention uses 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, the small number of modifying reagent molecules Combining a compound in which at least one element is labeled with a stable isotope and a compound having the same structure, but the aforementioned element is labeled with a stable isotope different from the above stable isotope Use. 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 A and B are prepared, the protein sample A is decorated with either a heavy reagent or a light reagent to obtain a modified protein sample A ′, and the protein sample B is heavy. The modified protein sample B ′ is obtained by modification with either the reagent or the light reagent. Similarly, when protein samples C and D are prepared, the protein sample C is modified with one of a heavy reagent and a light reagent to obtain a modified protein sample C ′, and the protein sample D is obtained with a heavy reagent and The modified protein sample D ′ is obtained by modifying with one of the light reagents. The modifying reagent that can be used in the present invention includes a compound having a structure represented by the general formula 1: R, 1 S—, represents an organic group, and X, represents a leaving group. R ₂ -X ₂ (R ₂ represents a substituted aralkyl group, X ₂ represents a leaving group), and the like. As the compound represented by the above general formula 1, that is, a sulfenyl compound, arylsulfenyl halide in which R in the above general formula 1 is a substituted aryl group is preferable. Further, as the arylsulfenyl halide, 2-nitrobenzenesulfenyl chloride (NBSCI) is preferable.

When using 2-nitrobenzene sulfoxide, as shown in the following formula: 2-Nitro [ ¹³ C ₆ ] benzenesulfuryl chloride (NBSCI heavy) as a heavy reagent and 2-nitro [ ¹² C ₆ ] benzenesulfuryl chloride (NBSCI light) as a light reagent Are preferably used.

Both NBSCI heavy reagent and NBSGI Light reagent are sold in the ¹³ GNBS ^(R) Stable Isotope Label ins Kit-N manufactured by Shimadzu Corporation.

2-Nitrobenzenesulfuric chloride (NBSGI)

Heavy reagents Light reagents In addition, sulfenyl compounds as modifying reagents for tributophane residues in protein peptides are described in detail in International Publication No. 2004/002950 Pamphlet.

As the compound represented by the general formula 2, benzyl halide is preferable. Furthermore, examples of benzyl halide include 2-hydroxy-5-nitrobenzyl bromide (2-hydroxy-5-η is robenzyl bromide).

When using the 2-hydroxy-one 5-nitrobenzyl blanking opening bromide, urchin by represented by the following formula, heavy reagent as 2-hydroxy one 5-nitro ^[13 C _6] benzyl pro bromide and 2-hydroxy-as light reagent It is preferable to use in combination with 5-nitro [ ¹² C ₆ ] benzylbutamide. 2-Hydroxy-5-nitrobenzene bromide

Light Reagents For benzyl halide compounds as modification reagents for tributophane residues in protein peptides, see Horton, HR and Koshland, D.E, Jr. (1972), Modification of proteins with active benzyl, ha I ides. , Methods in ENZY 0L0GY, 25, 468-482.

The modifying reagent is not limited to the compounds listed above, and a compound that has the ability to modify tritophanphan residues and does not have reactivity to oxidized tributophane residues can be combined with heavy and light reagents. There is no particular limitation.

(iii: Preparation of mixed sample for mass spectrometry)

Two kinds of modified protein samples A ′ and B ′ obtained by the above-mentioned modification step, or two kinds of modified protein samples C ′ and “'are mixed with each other. In a mixed sample for mass spectrometry, the mixed modified protein sample can be used for desalting, drying, resolubilization, reductive alkylation, fragmentation, and / or molecules with ribophane residues. It may be subjected to a concentration and separation treatment. The stage in which these treatments are performed may be before or after the mixing stage of the modified protein samples. However, those skilled in the art can appropriately determine. None of the methods for performing these processes is limited. For example, the desalting step can be performed by using a commonly used desalting column such as a Sephadex column. The resolubilization process can be performed in the same manner as the above-described solubilization process. The reductive alkylation treatment can also be performed by a usual method. Fragmentation treatment is not particularly limited, and digestion or chemical fragmentation with an enzyme such as rib ribsin, which is usually performed, can be performed. The concentration separation process is not particularly limited. For example, a Sephadex column or a column packed with a carrier having a phenyl group can be used. A column packed with a carrier having a phenyl group is useful when, for example, a 7Γ electronic compound (including NBSCI reagent) is used as the modifying reagent in the step (ii). Examples of such columns include Hi-Trap phenyl FF, Hi-Trap phenyl HP, Phenyl Sepharose 6 Fast Flow. Phenyl Sepharose High Performance, (above, Amersham Sciences), YC * GEL Ph (Made by YMC) Etc. Furthermore, the mixed sample for mass spectrometry may have been subjected to fractionation. Examples of the method for fractionation include a method using a system using a reverse phase column and HP LC. Each of the mixed samples for mass spectrometry when fractionation is performed will contain a plurality of fractions.

(iv: mass spectrometry)

Mass analysis is performed on the mixed sample for mass spectrometry. For measurement in the present invention A MALDI mass spectrometer can be used. In this case, a MALD-T0F type mass spectrometer (for example, AXIMA-GFRplus manufactured by Shimadzu Corporation / Kuraitos) or a MAID-IT-T0F type mass spectrometer (for example, AXIMA-QIT manufactured by Shimadzu Corporation / Kraitos) is used. When using an ALDI mass spectrometer, the matrix is not particularly limited. For example, DHB (2,5-dihydroxybenzoic acid; 2,5-dihydroxybenzo i G ac id), 4-CHCA

(—Cyanol 4-hydroxycinnamic acid) _N 3-CHCA (monocyan-3-hydroxycinnaic acid; a-cyano-3-hydroxycinnamic acid), 3H4NBA (3 —Hydroxy — 4-nitrobenzoic acid (3-hydroxy-4-nitrobenzoic acid) can be used as a matrix. When using a MALD 卜 T0F mass spectrometer, 4-GHCA should be used as a matrix. On the other hand, when a MAIDI-IT-T0F mass spectrometer is used, it is better to use 3-GHGA, 3H4NBA, a mixture of 3H4NBA and 4-GHGA as a matrix. Under these conditions, the mass spectrometric sample is measured with the mass spectrometer to obtain an MS spectrum. MS spectrum analysis is performed as follows.

For example, two protein samples of the same amount are prepared in the above step (i), and the above step (ii) using ¹³ G NBSGI and ¹² G NBSGI and the above step (Mi) including fragmentation are performed. Assume that a mixed sample for mass spectrometry is obtained. When at least one protein sample of the two protein samples prepared in the above step () is partially oxidized, that is, of the two protein samples, In at least one of the samples, a protein having an oxidized tributophan residue (hereinafter sometimes referred to as oxi-try-containing protein) is a protein having a non-oxidized tributophan residue (hereinafter referred to as non-oxi- Marked as Try-containing protein The resulting mixed sample for mass spectrometry contains a peptide having an NBS-modified tributophane residue (hereinafter sometimes referred to as an NBS-Try-containing peptide), A peptide having an oxidized tributophan residue (hereinafter sometimes referred to as an oxi-Try-containing peptide). NBS-Try containing peptides include ¹³ GNBS-Try containing peptides modified with heavy reagents and ¹² CNBS-Try containing peptides modified with light reagents.

In the analysis of the MS spectrum, attention is paid to the pair peak (ie, the peak of the BS-Try-containing peptide) having a mass-to-charge ratio corresponding to the molecular weight difference between the heavy reagent and the light reagent used in step (ii) above.

Since modification with NBS reagent occurs on the unoxidized tributophan residue, the intensity ratio of each of the two peaks that make up the paired peaks is in each of the two protein samples prepared in (i) above. This corresponds to the ratio of the amount of non-oxi-Try-containing protein contained in. Therefore, the reciprocal ratio of the intensity of the paired peaks corresponds to the ratio of the amount of protein containing οχί-Try.

In this way, in the MS analysis of the step (ίν), the degree of oxidation in the protein sample is obtained from the intensity ratio of each of the two peaks constituting the pair peak. In addition, the amount ratio of the protein sample prepared in step (i) may be the same as the intensity ratio of the pair peak in the S spectrum obtained in step (ίν). When such a spectrum is obtained when the oxidation numbers of both protein samples are unknown, oxi-try-containing protein is contained in either of the two protein samples in step (i). It is possible that the two protein samples contained the same percentage of oxi-Try containing protein. Therefore, in the MS analysis of step (iv), the pair peak corresponding to the NBS-Try containing peptide and the oxi-Try containing peptide having a structure corresponding to the NBS-Try containing peptide are included. It is preferable to confirm that a single peak has been detected. (Here, an oxi-Try-containing peptide having a structure corresponding to an rNBS-Try-containing peptide) means that the triftophan residue is converted to NBS. A peptide having the same structure as an NBS-Try-containing peptide except for the difference between being oxidized and oxidized). If such a single peak is detected, it can be determined that the two protein samples contained oxi-Try-containing protein at the same rate. In the following, the present invention will be described more specifically with reference examples.

[Reference Example 1]

Reference Example 1 shows an example in which protein sample A and protein sample B, in which the degree of oxidation of tribtophan residues are both unknown, are analyzed (see Fig. 1). First, prepare the same amount of protein sample A and protein sample B (i). Protein sample A is modified with ¹³ G NBS with NBSCI heavy reagent to obtain ¹³ C NBS-modified protein sample A ′. On the other hand, protein sample B is modified with ¹² CNBS with NBSGI light reagent to obtain ¹² G NBS-modified protein sample B ′ (ii). ¹³ G NBS-modified protein sample A 'and ¹² G NBS-modified protein sample B' are mixed / desalted, and reduced / alkylated, リブ ribucin digested, and concentrated to prepare a mixed sample for mass spectrometry (iii) . The obtained mixed sample for mass spectrometry is measured using a mass spectrometer (iv;). In the obtained MS vector, attention is paid to a pair peak having a mass difference of 6 Da corresponding to a molecular weight difference between NBSGI heavy reagent and NBSGI Mght reagent, that is, a peak of NBS-Try containing peptide. In Fig. 1, (ίν) shows a model of MS spectrum (horizontal axis: mass to charge ratio (Mass / Charge), vertical axis: relative intensity «Int.)). As shown in Figure 1, the ratio of the peak intensity of the ¹³ G NBS-Try-containing peptide derived from protein sample A to the peak intensity of the ¹² GNBS-Try-containing peptide derived from protein sample B of the paired peaks. But 3: 7. The same amount of protein sample A and protein sample B was used. From the above, it can be seen that the amount ratio of the protein containing ox i-Try between the protein sample A and the protein sample B was 7: 3. That is, in Reference Example 1, it can be seen that the relative ratio of the degree of tributophane residue oxidation between protein sample A and protein sample B was 7: 3.

[Consideration Example 2]

In Reference Example 2, protein sample C, in which the degree of oxidation of tryptophan residues is unknown, and non-oxidized protein sample D, in which it is known that tribtophan residues are not oxidized (ie, the degree of oxidation is 0%) An example of the case where is the analysis target is shown (see Fig. 2).

First, prepare the same amount of protein sample C and non-oxidized protein sample D (i). Protein sample C is ¹³ G NBS modified with NBSG I heavy reagent to obtain ¹³ G NBS modified protein sample A ′. On the other hand, non-oxidized protein sample D is ¹² G NBS-modified with NBSC I li ght reagent to obtain ¹² G NBS-modified protein sample B ′ (ii). Thereafter, the same steps (iii) and Uv) as in Reference Example 1 are performed. In the obtained MS spectrum, paying attention to the pair peak, the ¹³ G NBS-Try containing peptide derived from protein sample C against the peak intensity of the ¹² G NBS-Try containing peptide derived from non-oxidized protein sample D. Check the φ peak intensity. As shown in FIG. 2, when the intensity of the peak derived from the non-oxidized protein sample D is 1, it is assumed that the intensity of the peak derived from the protein sample C is 0.2. Since the same amount of the protein sample C and the non-oxidized protein sample D was used, it can be seen that in Reference Example 2, the acidity of tributophane residue of the protein sample C was 80%. The tributophane residue which is the target in the present invention is an amino acid belonging to a class with the lowest occurrence frequency in proteins. For example, the frequency In many cases, there is one triftophan residue per peptide molecule having a relatively short chain length, such as a peptide fragment containing a tributophan residue obtained by the fragmentation treatment in the step (iii).

In this case, in the present invention, the position of the oxidized tributophan residue can also be determined for a protein sample whose degree of oxidation has been clarified by MS analysis. Specifically, the amino acid sequence is determined by performing MS / MS measurement or more multi-stage MS measurement using at least one of the paired peaks obtained by MS measurement as a pretension ion. Determining the amino acid sequence is also specifying the position of the tributophan residue.

The paired peak obtained by MS itself is derived from a non-oxi-Try-containing protein in the protein sample. By knowing the position of the tributophane residue in the non-oxi-Try-containing protein, An oxi-Try-containing protein having a structure corresponding to the non-oxi-Try-containing protein (ie, the same as the non-oxi-Try-containing protein except for the difference in whether or not the tryptophan residue is oxidized) It is possible to know indirectly the oxidation position in the structure protein.

Example

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

[Example 1]

As a peptide to be analyzed, a delta sleep-inducing peptide (DS IP _: WAGGD ASGE (SEQ ID NO _: 1), m / z 848.1) was used. DS IP model sample in which 50 <½ tributophane residues are oxidized (ie, oxidized DS non-oxidized DS IP A sample containing an equal amount) was prepared, and the degree of oxidation of the model sample was quantified using 100% non-oxidized 03 IP as a control.

1. Preparation of oxidized DIP

1) Preparation of performic acid

900 L of formic acid and 100 L of hydrogen peroxide (3 Ow / w%) were mixed and reacted on ice for 2 hours.

2) Oxidation of DS I P

DS IP (manufactured by Peptide Institute) 100 was dissolved in 0.1 v / v% trifluoroacetic acid (TFA) aqueous solution 200 / L. To the resulting solution of D SIP, the formic acid 1OOL prepared in 1) above was added and reacted at room temperature for 2.5 hours. The disappearance of the raw materials was confirmed by liquid chromatography (LC), and then lyophilized.

2. Preparation of DS IP model sample in which 50% of tributophane residue was oxidized The total amount of DS IP oxidant prepared in 1. above was dissolved in 50 ju L of acetic acid to obtain a DS IP oxidant solution. Separately, non-oxidized D S I P I OOjW g was dissolved in 50 jU L of a 0.1 v / v% T F A aqueous solution to obtain a non-oxidized D S I P solution. A non-oxidized DS I P solution was added to and mixed with the DS I P oxide solution. The resulting mixture corresponds to a sample containing 50% tryptophan residue oxidized DIP200 / ig in a mixed solution of acetic acid and 0.1 v / v% TFA in water (volume ratio 1: 1). .

3. NBS reagent ^{^{(13 G NBS (R) Stable}} Isotope Labeling it-N, manufactured by Shimadzu Corporation) by quantitative oxidation of the Toributofan residues in the model sample

200 g of non-oxidized DS I P was dissolved in a mixed solution of acetic acid—0 · 1 v / v% TFA aqueous solution (volume ratio 1: 1) to obtain a non-oxidized DS IP sample. For non-oxidized DS IP samples, ^A solution of ¹² C NBS reagent (1 mg) in acetic acid (1 0 OL) was added. On the other hand, a solution of ¹³ C NBS reagent (1 mg) in acetic acid (1 00 L) was added to the DS IP model sample prepared in 2 above. Both samples were allowed to react for 3 hours at room temperature. After completion of the reaction, 2 L of each aliquot was taken from both samples and mixed with each other, and further diluted with 200 v L of 0.1 v / v% TFA aqueous solution. After Z ip T ip treatment, MA LDI—TO F MS was measured using a mass spectrometer (AXIMA-GFR, manufactured by Shimadzu Corporation) using α-cyan-4-hydroxycinnamic acid (CHCA) as a matrix. It was measured.

4. MA L D I—TO F MS results

On the MS spectrum, paired peaks of D S IP modified with NBS reagent were observed at m / z 1002.58 and 1008.64. The area ratio of each peak is

Area (1002.58): Area (1008.64) = 1.99: 1.

This is a sample whose peak with a larger mass-to-charge ratio is modified with ¹³ C NBS reagent, that is, a DS IP model sample with oxidized tributophane residues, and the degree of oxidation of tributophane residues in the model sample is 50%. It is shown that. This result was in good agreement with the DS IP model sample containing 50% DSIP with oxidized tribtophan residues and 50% DS IP with no tryptophan residues oxidized. This indicates that the oxidation degree of tributophan residue in the sample can be obtained from the intensity of the pair peak in the MS spectrum. Therefore, it was shown that the relative or absolute oxidation degree can be obtained from the intensity of the paired peak even if the oxidation degree of at least one of the two types of samples is unknown.

In the above example, the protein having a tributofan residue oxidation degree of 50% It was confirmed that the effect of the present invention was achieved by artificially preparing a sample and analyzing this sample. Therefore, the present invention can naturally be carried out on a protein sample having a natural degree of tributophane residue oxidation. In the above examples, the analysis was performed using the DSIP sample as the protein sample to be analyzed and using the NBS reagent as a modification reagent for the isotope labeling method. However, the present invention is also applicable to protein samples other than DSIP samples and isotope labeling modifying reagents other than NBS reagents. For this reason, the above-described embodiment is merely an example in all respects and should not be interpreted in a limited manner. 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) Prepare a protein sample A in which the degree of oxidation of tributophane residues is unknown and a protein sample B in which the degree of oxidation of tryptophan residues is unknown.

'(i i) As a modifying reagent, two compounds having the same molecular structure and different molecular weights by containing isotopes having different mass numbers are prepared.

The protein sample A is modified with tributophan residue using either one of the two kinds of compounds to obtain a modified protein sample A ′, and separately for the protein sample B, Tributofan residue is modified using the other of the two compounds to obtain a modified protein sample B ′,

(ii) Mixing the modified protein sample A ′ and the modified protein sample B ′ to obtain a mixed sample for mass spectrometry,

(iv) subjecting the mixed sample for mass spectrometry to mass spectrometry and quantifying the degree of oxidation of the tributophane residue in each of the protein sample A and the protein sample B, and the oxidized tryptophan residue in the protein To perform quantitative determination.

2. The method for quantifying an oxidized tributophan residue in a protein according to claim 1, wherein the modifying reagent is a sulfenyl compound.

3. The method for quantifying an oxidized tributophan residue in a protein according to claim 2, wherein the sulfinyl compound is aryl sulfonyl chloride.

4. The arylsulfenyl halide is 2-nitrobenzenesulfuric acid. The method for quantifying an oxidized tributophan residue in a protein according to claim 3, which is a chloride.

5. Determine the position of the oxidized tributophan residue in the protein by performing the method according to any one of claims 1 to 4 and then performing amino acid sequencing. Method.

6. (i) Prepare protein sample C for which the degree of oxidation of tributofan residues should be measured, and 卜 protein sample D for which the degree of oxidation of ribtophan residues is known,

(ii) Two types of compounds having different molecular weights by containing isotopes having the same molecular structure and different mass numbers as the modifying reagent are prepared. Tributofan residue is modified using one of the compounds of the above to obtain a modified protein sample C ′, and separately from the protein sample D, either of the two compounds is used. The compound is used to modify the tryptophan residue to obtain a modified protein sample D ′,

(ii) Mixing the modified protein sample C ′ and the modified protein sample D ′ to obtain a mixed sample for mass spectrometry,

(iv) Quantifying the oxidized tributophane residue in the protein, comprising quantifying the degree of oxidation of the tributophane residue in the protein sample C by subjecting the mixed sample for mass spectrometry to mass spectrometry How to do.

7. The method for quantifying an oxidized tributophan residue in a protein according to claim 6, wherein the modifying reagent is a sulfonyl compound.

8. The method for quantifying an oxidized tryptophan residue in a protein according to claim 7, wherein the sulfenyl compound is aryl sulfenyl halide.

9. The method for quantifying oxidized tributophane residues in a protein according to claim 8, wherein the arylsulfuryl chloride is 2-nitrobenzenesulfuryl chloride.

10. A method for determining the position of an oxidized tributophan residue in a protein by performing the method according to any one of claims 6 to 9 and then performing amino acid sequencing.