JP4609370B2 - Methods for treating sulfur-containing peptides and methods for analyzing sulfur-containing peptides - Google Patents

Description

  The present invention relates to protein / peptide chemistry and mass spectrometry. More particularly, the present invention relates to mass spectrometry of proteins containing sulfur atoms.

  Oxidizing amino acid residues that have readily oxidizable immediate chains in proteins and peptides and examining their structural and biological activity changes is one of the major research fields of protein and peptide chemistry, and has been active for a long time. Research has been conducted. What is important when performing the oxidation reaction is to selectively oxidize only the target residue. As the oxidizing agent, ozone, N-chlorosuccinimide, N-bromosuccinimide, chloramine T, trichloromethanesulfonyl chloride, hydrogen peroxide and the like are used. A method of performing photooxidation using methylene blue as a photosensitizer is also known.

Furthermore, the peptide containing methionine is oxidized to methionine sulfoxide by allowing hydrogen peroxide to act, or to methionine sulfone by allowing hydrogen peroxide and formic acid to act, and the resulting oxidant is FAB-CID-MS / By using the MS measurement, it has been found that metastable ions in which the CH ₃ SOH moiety is removed from the sulfoxide and the CH ₃ SO ₂ moiety is removed from the sulfone are easily generated (for example, see Non-Patent Document 1). .

  On the other hand, as a protein / peptide analysis method, an NBS method is known in which isotope labeling is performed using 2-nitrobenzenesulfenyl chloride (NBSCl) and differential display is performed using a mass spectrometer (for example, patents). Reference 1 and Non-Patent Document 2).

International Publication No. 2004/002950 Pamphlet F. M. Lagerwerf et al., "Rapid Communications In Mass Spectrometry", 1996, Vol. 10, p. 1905-1910 Hiroki Kuyama, Makoto Watanabe, Chikako Toda, Eiji Ando, Koichi Tanaka and Osamu Nishimura, “Rapid Communications in. “Rapid Communications In Mass Spectrometry”, 2003, Vol. 17, p. 1642-1650

  When preparing protein / peptide samples, amino acid residues having side chains that are easily oxidized are often partially oxidized in the sample. Such a protein / peptide sample should be seen as a single species peak in the original unoxidized state when it is fragmented into peptide fragments and measured with a mass spectrometer, for example. It will be observed dispersed. This means a decrease in sensitivity of the peptide fragment and is an undesirable phenomenon. Furthermore, in the relative quantitative analysis of proteins by the NBS method, not only the sensitivity is lowered, but also the oxidant is mixed, the quantitative accuracy is lowered.

  In order to solve this problem, it is possible to converge to a single chemical species in either case by reducing oxidized proteins / peptides or oxidizing unoxidized proteins / peptides. Conceivable. However, in the method of converging chemical species as a reductant, in consideration of the reactivity that causes oxidation during sample preparation, on the mass spectrum, the peak of the peptide fragment of interest is that of the oxidant but not oxidized. It is easily predicted to be observed with the body. Conversely, in the method of converging chemical species as an oxidant, the peak of the target peptide fragment can be predicted to be observed as a single peak because the oxidant is chemically stable.

  The present inventors focus on methionine residues in proteins / peptides and selectively oxidize sulfur atoms of methionine residues to converge methionine-containing chemical species in protein / peptide samples as methionine oxidants. Aimed at that. As described in the above prior art, various reagents are known for the oxidizing agent. As for selective oxidation of the sulfur atom of the methionine residue in the target protein / peptide, hydrogen peroxide, N- It has been reported that this is possible with reagents such as chlorosuccinimide, chloramine T, and methylene blue used as a photosensitizer in photooxidation. However, when N-chlorosuccinimide, chloramine T, or methylene blue is used, delicate conditions must be set, and separation and purification after the reaction may be troublesome.

Another object of the present invention is to apply such a method for oxidizing a methionine residue to analysis of a protein / peptide using mass spectrometry. Therefore, it is better that the time required for the treatment process after the oxidation reaction is as small as possible. In this way, looking at the oxidizing agent, hydrogen peroxide seems to be suitable.
In addition, there is no example in which an oxidation reaction is used for a protein / peptide in order to achieve improvement in sensitivity and quantification of a protein / peptide analysis method using a mass spectrometer.

An object of the present invention is to provide a method for selectively oxidizing only a sulfur atom of a methionine residue in a protein or peptide containing a sulfur atom.
Another object of the present invention is to provide a method for selectively converging a methionine residue-containing chemical species as an oxidant in a protein / peptide sample in which oxidized methionine residues and non-oxidized methionine residues can coexist. . A further object of the present invention is to provide a method capable of improving the sensitivity and quantitativeness of protein / peptide analysis using mass spectrometry.

  The present invention includes the following inventions. Hereinafter, in the present invention, a peptide is used in the meaning including oligopeptide, polypeptide and protein.

The following [1] to [7] are directed to a method for treating a sulfur atom-containing peptide.
[1]
To a sample containing a peptide (A) having an S-alkylated cysteine residue and / or a sulfenylated tryptophan residue and a methionine residue, hydrogen peroxide is added as an oxidation step to add a methionine residue. The processing method of a sulfur atom containing peptide including performing the process of oxidizing only the sulfur atom of.

  The following [2] is a method of selectively oxidizing a methionine residue-containing peptide species by selectively oxidizing a sulfur atom of a methionine residue in a protein / peptide sample in which an oxidized methionine residue and a non-oxidized methionine residue are mixed. Is directed to a converged form.

[2]
The sample containing the peptide (A) having the S-alkylated cysteine residue and / or the sulfenylated tryptophan residue and the methionine residue is obtained from an S-alkylated cysteine residue and / or A peptide (A-oxide) having a sulfenylated tryptophan residue and an oxidized methionine residue;
By performing the oxidation step, the peptide (A) in the sample is converted into an S-alkylated cysteine residue and / or a sulfenylated tryptophan residue having the same structure as the peptide (A-oxide). The method for treating a sulfur-containing peptide according to [1], wherein the peptide is converted to a peptide having a group and an oxidized methionine residue.

  The following [3] is directed to a form obtained by treating the sample containing the oxidized methionine residue and the non-oxidized methionine residue in [2] above with respect to the cysteine residue and / or tryptophan residue. In this case, the oxidized methionine residue is not generated by aggressive oxidation treatment, but preferably is generated as a by-product in the treatment of cysteine residue and / or tryptophan residue.

[3]
The sample containing the peptide (A) and the peptide (A-oxide) further comprises the following steps:
To a peptide having a cysteine residue and / or tryptophan residue and a methionine residue,
If having the cysteine residue, performing S-alkylation of the cysteine residue, and / or
When having the tryptophan residue, sulfenylation of the tryptophan residue;
By performing processing including
Peptide (A) having the S-alkylated cysteine residue and / or sulfenylated tryptophan residue and methionine residue as main products,
Obtained as a by-product is a step of obtaining a peptide (A-oxide) having the S-alkylated cysteine residue and / or sulfenylated tryptophan residue and an oxidized methionine residue. The processing method of the sulfur containing peptide as described in [2] which is a thing.

[4]
The method for treating a sulfur atom-containing peptide according to any one of [1] to [3], wherein the S-alkylated cysteine residue is an S-carbamidomethylated cysteine residue.

[5]
The method for treating a sulfur atom-containing peptide according to any one of [1] to [4], wherein the sulfenylated tryptophan residue is a 2-nitrobenzenesulfenylated tryptophan residue.

[6]
The method for treating a sulfur atom-containing peptide according to [5], wherein the 2-nitrobenzenesulfenylated tryptophan residue is obtained by allowing 2-nitrobenzenesulfenyl chloride to act on a tryptophan residue.

[7]
The method for treating a sulfur atom-containing peptide according to any one of [1] to [6], wherein the sulfur atom of the methionine residue is oxidized to sulfoxide.

  The invention [8] below is directed to protein mass spectrometry. By using the method according to any one of [1] to [7] above, the methionine residue-containing peptide species is converged as an oxidant, so that the peptide species can be detected with high sensitivity and quantitative properties in the mass spectrum. Is done.

[8]
Oxidized with an S-alkylated cysteine residue and / or a sulfenylated tryptophan residue obtained by the method for treating a sulfur atom-containing peptide according to any one of [1] to [7] A method for analyzing a sulfur atom-containing peptide, comprising measuring a peptide having a methionine residue using a mass spectrometer.

  According to the present invention, it is possible to provide a method for selectively oxidizing only the sulfur atom of a methionine residue in a protein or peptide containing a sulfur atom.

  Further, according to the present invention, it is possible to provide a method for selectively converging a methionine residue-containing chemical species as an oxidant in a protein / peptide sample in which an oxidized methionine residue and a non-oxidized methionine residue can be mixed. Furthermore, according to the present invention, it is possible to provide a method capable of improving the sensitivity and quantitativeness of protein / peptide analysis using mass spectrometry.

  More specifically, the peak of methionine residue-containing peptides has been detected in the past due to the influence of the presence of an oxidant generated as a by-product in the peptide treatment for preparing a mass spectrometry sample. Since all methionine residues are converged as oxidants, the peak of the methionine residue-containing peptide can be detected as a single peak. In addition, in mass spectrometry, an oxidized form of methionine is more susceptible to cleavage than a non-oxidized form, so that more structural information can be obtained in PSD (post-source decay) measurement or multistage measurement of MS squared or more. .

Hereinafter, proteins or peptides are collectively referred to as peptides.
<Treatment of sulfur atom-containing peptides>
In this invention, the processing method of a sulfur atom containing peptide is provided. In the method for treating a sulfur atom-containing peptide of the present invention, only a sulfur atom of a methionine residue is selectively oxidized in a peptide containing a sulfur atom. More specifically, a protein or peptide containing a sulfur atom contains a cysteine residue or an S-modified cysteine residue, a sulfenylated tryptophan residue, or a methionine residue as a sulfur atom-containing amino acid residue. .

  The present invention can be used for the purpose of selectively oxidizing a methionine residue in a peptide, for example, when a methionine residue in a peptide is oxidized to examine changes in its structure and biological activity.

  The present invention includes a form in which an oxidation step is performed on a sample containing a peptide (A) having an S-alkylated cysteine residue and / or a sulfenylated tryptophan residue and a methionine residue. (Formula 1 below). In the oxidation step, only the sulfur atom of the methionine residue is oxidized by the action of hydrogen peroxide.

In the following formula 1, a carboxymethylated cysteine residue is described as an example of an S-alkylated cysteine residue, and a nitrobenzenesulfenylated tryptophan residue is described as an example of a sulfenylated tryptophan residue. Has been. In the following formula 1, C is a cysteine residue, C (CAM) is a carboxymethylated cysteine residue, W is a tryptophan residue, W (NBS) is a nitrobenzenesulfenylated tryptophan residue, and M is oxidized Not a methionine residue, M ^* represents an oxidized methionine residue, and a dotted line represents a peptide chain.

  Therefore, in the following formula 1, a peptide having a carboxymethylated cysteine residue and a methionine residue; a peptide having a nitrobenzenesulfenylated tryptophan residue and a methionine residue; and carboxymethylated It has been shown that three types of peptides having a cysteine residue, a nitrobenzenesulfenylated tryptophan residue, and a methionine residue were each converted by an oxidation reaction using hydrogen peroxide.

  Further, the present invention, for example, when the peptide to be analyzed is appropriately treated as a sample for measurement / analysis, the peptide is exposed to an environment susceptible to oxidation of the methionine residue, and the methionine residue moiety It can be used for the purpose of selective and complete oxidation of in-residues to the eye thio when the subsequent oxidation or the like hinders subsequent measurement and analysis.

  In the present invention, a sample comprising the peptide (A) having the S-alkylated cysteine residue and / or the sulfenylated tryptophan residue represented by the above formula 1 and a methionine residue, It further includes a peptide (A-oxide) which is an oxidized form of peptide (A) and has an S-alkylated cysteine residue and / or a sulfenylated tryptophan residue and an oxidized methionine residue. In some cases, a mode in which an oxidation process is performed on the sample is included (Formula 2 below).

  More practically, in the present invention, a sample containing the peptide (A) and the peptide (A-oxide) represented by the following formula 2 contains a cysteine residue and / or a tryptophan residue and a methionine residue. The sample containing the peptide having the form includes a form obtained by performing a treatment step including S-alkylation of a cysteine residue and / or sulfenylation of a tryptophan residue (Formula 3 below). In this case, the treatment including S-alkylation and / or sulfenylation yields peptide (A) as the main product and peptide (A-oxide) as the by-product.

As shown in the above formulas 2 and 3, the sample methionine residue-containing peptide species is converged as an oxidant after the oxidation step.
Hereinafter, the treatment step including S-alkylation and / or sulfenylation (that is, accompanied by partial oxidation of methionine residue) is referred to as step (1), and the oxidation step is referred to as step (2). The invention will be further described.

(Step 1: Peptide treatment step that may involve partial oxidation of methionine residue)
In the present invention, a peptide having a cysteine residue and / or a tryptophan residue and a methionine residue can be used as a target peptide. In this case, among the amino acid residues of the target peptide, the methionine residue and the cysteine residue are sulfur atom-containing amino acid residues, and the tryptophan residue is treated with a sulfenyl compound by the NBS method or the like. , A sulfur atom-containing amino acid residue (sulfenylated tryptophan residue).

The present invention is particularly useful when the target peptide has a tryptophan residue and the treatment with a sulfenyl compound in which the tryptophan residue has a specific modification ability is performed.
On the other hand, when the target peptide has a cysteine residue, the sulfhydryl group of cysteine is modified with an appropriate alkyl group which may be substituted. Although it does not specifically limit as a modification form by the alkyl group which may be substituted, Carbamidomethylation which is a technique well-known to those skilled in the art is common.

The sulfenyl compound is not particularly limited as long as it can specifically modify the tryptophan residue in the peptide, but preferred is 2-nitrobenzenesulfenyl chloride (NBSCl; NBS). Reagent). When performing differential display using mass spectrometry (that is, when performing the NBS method), 2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride (NBSCl heavy) as a heavy reagent and 2-nitro [ ¹² C as a light reagent. ₆ ] Benzenesulfenyl chloride (NBSCl light) can be used in combination. NBSCl heavy reagent and NBSCl light reagent are both sold in the ¹³ C NBS ^(R) Stable Isotope Labeling Kit-N manufactured by Shimadzu Corporation.

  The sulfenyl compound having the ability to specifically modify the tryptophan residue in the peptide is described in detail in International Publication No. 2004/002950 pamphlet and the like.

The modification method using a sulfenyl compound is not particularly limited. For example, when NBSCl is used, the following method is given as a preferred example.
The NBSCl 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 NBSCl reagent can be used in a large excess of about 20 times equivalent to the peptide 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 peptide samples.
The modification reaction can be performed by adding NBSCl reagent to a peptide 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.

  In tryptophan modification with the sulfenyl compound, methionine residues in peptides are often partially oxidized. Similarly, in the S-alkylation of cysteine residues, partial oxidation of methionine residues may occur. By treatments other than those described above, that is, various treatments usually performed on peptides, methionine residues in peptides are often partially oxidized.

  Specifically, the above-mentioned sulfenylation and / or S-alkylation and other treatments that can be performed on the peptide produce a peptide mixture containing the peptide (A) and its oxidized form (A-oxide). That is, a peptide (A) having an S-alkylated cysteine residue and / or a sulfenylated tryptophan residue and a methionine residue is produced as the main product. A peptide (A-oxide) having an S-alkylated cysteine residue and / or a sulfenylated tryptophan residue and an oxidized methionine residue as a by-product together with this main product. Arise.

  When the peptide mixture is measured using, for example, a mass spectrometer as in the past, the peptide (A) to be detected is detected together with its oxidant (A-oxide). Although it is derived from the same sequence in the original peptide, it is dispersed and detected in the peak of peptide (A) and the peak of its oxidant (A-oxide), which means the sensitivity of the peptide to be detected. It will cause a decrease and a decrease in quantitativeness.

(Process 2: Selective / complete oxidation process of sulfur atom of methionine residue)
Only the sulfur atom of the methionine residue is oxidized among the sulfur atoms in the peptide (1) in the peptide mixture obtained in the step (1). In the present invention, an oxidation reaction using hydrogen peroxide as an oxidizing agent is performed. In the oxidation reaction of the present invention, only the sulfur atom of the methionine residue among the sulfur atoms in the peptide (A) is oxidized, and the sulfur atom of the cysteine residue that has been S-alkylated and the tryptophan residue that has been sulfenylated. Sulfur atoms are not oxidized. As a result, the peptide having the same structure as the peptide (A-oxide) as a by-product generated in the step (1), that is, the S-alkylated cysteine residue and / or the sulfenylated tryptophan residue A peptide having a group and an oxidized methionine residue is produced. Thus, in the step (1), the peptide species that existed in different forms of the oxidized methionine residue and the non-oxidized methionine residue, despite being derived from the same sequence in the original peptide. Converges as an oxidized methionine residue.

  In addition, the methionine oxidant (A-oxide) in the peptide mixture obtained in the step (1) is usually sulfoxide. Therefore, the oxidation treatment in this step is usually performed under conditions for converting the thioether group of the methionine residue to a sulfinyl group.

Specifically, the oxidation reaction in this step is performed by allowing hydrogen peroxide to act as an oxidizing agent on the peptide mixture. The use of hydrogen peroxide not only enables oxidation of only the sulfur atom of the methionine residue in the peptide mixture obtained in the above step (1), but also facilitates the setting of conditions and the number of processing steps after the reaction. From the point of view, it is preferable. Hydrogen peroxide can be used as an aqueous solution, and the concentration and amount of hydrogen peroxide water can be appropriately selected by those skilled in the art. For example, it can be used so that hydrogen peroxide is 1/100 to 1/6 times the amount of the target protein on a weight basis.
The temperature of the oxidation reaction when hydrogen peroxide is used is preferably 15 ° C. to 30 ° C., for example. The reaction time can be 10 minutes to 1 hour.

  When the sample after the oxidation reaction is measured by a mass spectrometer, the oxidation treatment using hydrogen peroxide may be performed on the MS plate. In this case, first, the peptide mixture obtained in the step (1) may be dropped on the MS plate, and then hydrogen peroxide solution may be further dropped on the dropped peptide mixture. After finishing the oxidation reaction, mass spectrometry can be performed directly by the MS plate. Thus, it is preferable to use hydrogen peroxide as the oxidizing agent in that the number of treatment steps after the reaction is small.

<Method for analyzing sulfur atom-containing peptides>
The method for treating a sulfur atom-containing peptide of the present invention is particularly useful when a peptide sample obtained thereby is measured using a mass spectrometer.
Therefore, the present invention further provides a method for analyzing a sulfur atom-containing peptide. Conventionally, peaks of methionine residue-containing peptides have been detected in a dispersed manner due to the presence of an oxidant generated as a by-product in peptide processing to prepare a mass spectrometry sample. Since the residue is converged as an oxidant, the peak of the methionine residue-containing peptide can be detected as a single peak. Therefore, the present invention has an effect of improving the sensitivity of analysis and the accuracy of quantification as compared with the conventional method.

  Converting a methionine residue to an oxidized methionine residue further has the following advantages. That is, a peptide in which a sulfur atom is oxidized is more susceptible to cleavage in mass spectrometry than a non-oxidized form. Therefore, PSD (post-source decay) measurement can obtain more structural information in multi-step measurement of the square of MS or more. Also in this respect, the present invention has an effect of improving the accuracy of analysis as compared with the conventional method.

As a specific analysis protocol, the NBS method is preferably used. An example of the NBS method to which the method of the present invention is applied will be introduced below.
<1> Prepare two kinds of protein samples, ie, a protein sample I to be analyzed and a control protein sample II thereof.
<2> The protein sample I is modified with any one of 2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride and 2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride, and separately the protein sample II Is modified with either one of 2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride and 2-nitro [ ¹² C ₆ ] benzenesulfenyl chloride.
<3> Mix the modified protein sample I and the modified protein sample II.
<4> Remove unreacted reagents using a desalting column.
<5> Perform reductive alkylation treatment.
<6> The obtained modified protein mixture is reduced and alkylated, and then digested into a peptide mixture containing a modified peptide fragment and an unmodified peptide fragment.
<7> An oxidation treatment using hydrogen peroxide is performed on the peptide mixture.
<8> The modified peptide fragment is concentrated and separated from the peptide mixture.
<9> Perform mass spectrometry.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

[Example 1]
Neuromedin C (GNHWAVGHLM-NH _2: SEQ ID NO: 1, M-NH ₂ represents an amidated methionine residue) was a peptide sample to be analyze. 100 micrograms of this peptide sample was dissolved in 50 microliters of a 0.1 v / v% TFA aqueous solution. A solution obtained by dissolving 0.3 mg of NBS reagent (2-nitrobenzenesulfenyl chloride; ¹² C-6) in 50 microliters of acetic acid was added to the obtained peptide solution and reacted at room temperature for 1 hour. After the reaction, desalting was performed with a column of LH20, followed by concentration under reduced pressure to obtain a NBS-modified Neuromedin C sample.
To this NBS-modified Neuromedin C sample, 300 microliters of 0.1 v / v% TFA aqueous solution was added and dissolved. 100 microliters of that was taken and 1 microliter of 30 v / v% hydrogen peroxide water was added. When the reaction was performed at room temperature and the reaction was monitored after 30 minutes and 1 hour, the oxidation reaction converged in 1 hour.

  The NBS converted Neuromedin C sample before the oxidation reaction and the oxidized / NBS converted Neuromedin C sample after the oxidation reaction were measured using a mass spectrometer AXIMA-FCR (manufactured by Shimadzu Corporation). The mass spectrum of the NBS-modified Neuromedin C sample is shown in FIG. 1 (a), and the mass spectrum of the oxidized / NBS-modified Neuromedin C sample is shown in FIG. 1 (b). In all the figures, the horizontal axis represents the mass-to-charge ratio (Mass / Charge), and the vertical axis represents the relative intensity.

In FIG. 1A, a peak 16 Da larger than the peak is detected together with the peak of the NBS-modified Neuromedin C molecule. From this, it can be said that such an +16 Da peak was detected because an oxidized form of NBS-modified Neuromedin C (that is, oxidized / NBS-modified Neuromedin C molecule) was generated as a by-product in the preparation of NBS-modified Neuromedin C sample.
In FIG. 1 (b), it can be seen that the peak of the NBS-modified Neuromedin C molecule detected in FIG. 1 (a) disappears and is converged to the oxidized / NBS-modified Neuromedin C molecule and detected. Further, it was found that the sulfur atom of the NBS-modified tryptophan residue was not oxidized by the oxidation reaction, and only the sulfur atom of the methionine residue was selectively oxidized.

[Example 2]
Egg white lysozyme was used as a protein sample to be analyzed. 100 micrograms of this protein sample was dissolved in 25 microliters of 6M guanidine hydrochloride. A solution obtained by dissolving 0.2 mg of NBS reagent (2-nitrobenzenesulfenyl chloride; ¹² C-6) in 25 μl of acetic acid was added to the obtained protein solution and reacted at room temperature for 1 hour. After the reaction, the solution was desalted with an LH20 column and then concentrated under reduced pressure. Tris-HCl buffer was added to the concentrated residue for dissolution, and reduction with TCEP and alkylation with iodoacetamide were performed. Subsequently, trypsin solution (Trypsin 6.7 microgram / buffer 300 microliter) was added and digestion (37 ° C., 14 hours) to obtain a digested NBS egg white lysozyme.

  Measurement of NBS-modified egg white lysozyme digest before oxidation and oxidation / NBS-modified egg white lysozyme digest after oxidation using mass spectrometer AXIMA-FCR (manufactured by Shimadzu Corporation) went. Among the obtained mass spectra, the range of mass to charge ratio of 1977-2030 is shown in FIG. 2, and the range of 1295-1320 is shown in FIG.

In FIG. 2 (a), an NBS-modified peptide fragment (IVSDGNGMNAW (NBS) VAW (NBS) R: SEQ ID NO: 2, W (NBS) represents an NBS-modified tryptophan residue contained in an NBS-modified egg white lysozyme digest. A peak that is 16 Da larger than the peak is detected. From this, it can be said that such a +16 Da peak was detected because an oxidized form of the NBS-modified peptide fragment (oxidized / NBS-modified peptide fragment) was generated as a by-product in the preparation of NBS-modified egg white lysozyme digest. .
In FIG. 2 (b), it can be seen that the peaks of the NBS-modified peptide fragments detected in FIG. 2 (a) disappeared and converged to the corresponding oxidized / NBS-modified peptide fragments were detected. Further, it was found that the sulfur atom of the NBS-modified tryptophan residue was not oxidized by the oxidation reaction, and only the sulfur atom of the methionine residue was selectively oxidized.

  In FIG. 3 (a), a peptide fragment without a methionine residue (W (NBS) W (NBS) C (CAM) NDGR: SEQ ID NO: 3, W (NBS)) contained in the digested egg white lysozyme of NBS Are NBS-modified tryptophan residues, and C (CAM) is an S-carbamidomethylated cysteine residue). In this fragment, no change in molecular weight was observed before and after the oxidation reaction. From this, it was confirmed that the sulfur atom of the NBS-modified tryptophan residue and the sulfur atom of the carbamidomethylated cysteine were not oxidized.

The mass-spectrometry result of Neuromedin C obtained in Example 1 is shown. The mass spectrometry result of Lysozyme obtained in Example 2 is shown. The mass spectrometry result of Lysozyme obtained in Example 2 is shown.

Claims (8)

  To a sample containing a peptide (A) having an S-alkylated cysteine residue and / or a sulfenylated tryptophan residue and a methionine residue, hydrogen peroxide is added as an oxidation step to add a methionine residue. The processing method of a sulfur atom containing peptide including performing the process of oxidizing only the sulfur atom of. The sample containing the peptide (A) having the S-alkylated cysteine residue and / or the sulfenylated tryptophan residue and the methionine residue is obtained from an S-alkylated cysteine residue and / or A peptide (A-oxide) having a sulfenylated tryptophan residue and an oxidized methionine residue;
By performing the oxidation step, the peptide (A) in the sample is converted into an S-alkylated cysteine residue and / or a sulfenylated tryptophan residue having the same structure as the peptide (A-oxide). The method for treating a sulfur-containing peptide according to claim 1, wherein the peptide is converted to a peptide having a group and an oxidized methionine residue. The sample containing the peptide (A) and the peptide (A-oxide) is prepared by the following process:
In a sample containing a peptide having a cysteine residue and / or a tryptophan residue and a methionine residue,
If having the cysteine residue, performing S-alkylation of the cysteine residue, and / or
When having the tryptophan residue, sulfenylation of the tryptophan residue;
By performing processing including
Peptide (A) having the S-alkylated cysteine residue and / or sulfenylated tryptophan residue and methionine residue as main products,
Obtained as a by-product is a step of obtaining a peptide (A-oxide) having the S-alkylated cysteine residue and / or sulfenylated tryptophan residue and an oxidized methionine residue. The processing method of the sulfur containing peptide of Claim 2 which is a thing.   The method for treating a sulfur atom-containing peptide according to any one of claims 1 to 3, wherein the S-alkylated cysteine residue is an S-carbamidomethylated cysteine residue.   The method for treating a sulfur atom-containing peptide according to any one of claims 1 to 4, wherein the sulfenylated tryptophan residue is a 2-nitrobenzenesulfenylated tryptophan residue.   The method for treating a sulfur atom-containing peptide according to claim 5, wherein the 2-nitrobenzenesulfenylated tryptophan residue is obtained by allowing 2-nitrobenzenesulfenyl chloride to act on a tryptophan residue.   The sulfur atom-containing peptide treatment method according to claim 1, wherein the sulfur atom of the methionine residue is oxidized to sulfoxide.   Oxidized with an S-alkylated cysteine residue and / or a sulfenylated tryptophan residue obtained by the method for treating a sulfur atom-containing peptide according to any one of claims 1 to 7. A method for analyzing a sulfur atom-containing peptide, comprising measuring a peptide having a methionine residue using a mass spectrometer.

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