JP4656236B2 - Method for quantifying and locating oxidized tryptophan residues in proteins - Google Patents

Description

  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 quantifying and positioning oxidized tryptophan residues in a protein (Method for Quantification and Positioning of Oxidized Tryptophan Residue in Protein).

Conventionally, quantification of oxidized tryptophan residues has been carried out by measuring UV-visible spectra and analyzing changes in absorbance due to oxidation (for example, WE Savige and A. Fontana, International Journal of Peptide and Protein). Research, 15, 285 (1980)).
On the other hand, as a substance capable of selectively modifying tryptophan residues in proteins and peptides, sulfenyl halide (for example, Scoffone E, Fontana A, Rocchi R, Sulfenyl Halides as Modifying Reagents for Polypeptides. Proteins. Proteins. of Tryptophan Residues, Biochemistry, 7 (1968) 971-979) and benzyl halides (e.g. Horton, HR and Koshland, DE, Jr. (1972), Modification of proteins with activities in the United States). in ENZYM LOGY, 25,468-482 reference), and the like are known.
Quantitative proteomic analysis focusing on tryptophan residues in proteins and peptides has been performed using a stable isotope-labeled compound of sulfenyl halide (for example, Rapid Communications in Mass Spectrometry, 17, 1642-1650 (2003). ), International Publication No. 2004/002950 pamphlet).
W. E. WE Savige and A.S. A. Fontana, “International Journal of Peptide and Protein Research”, 1980, Vol. 15, p. 285 Scofone E, Fontana A, and Rocchi R, “Biochemistry”, 1968, vol. 7, p. 971-979 Horton, HR and Koshland, DE, Jr., “Methods in ENZYMOLOGY”, 1972, 25th. Volume, p. 468-482 “Rapid Communications in Mass Spectrometry” 2003, Vol. 17, p. 1642-1650 International Publication No. 2004/002950 Pamphlet

Object of the Invention Oxidation of a protein occurring in a living body causes reduction in protein activity, expression of toxicity, and the like, and it has been reported that it is associated with cataract, 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 in vivo or in vitro conditions is considered to have great significance in elucidating life phenomena and structure-activity relationships.
In the present invention, attention is focused on the degree of oxidation of tryptophan residues. Tryptophan residues are amino acid residues that are easily oxidized along with methionine residues, and thus are attracting attention in studies such as oxidative stress and age-related changes.
Among the conventional techniques listed above, the method of quantifying oxidized tryptophan residues using an ultraviolet-visible spectrum cannot perform accurate quantification. Also, it is not known which tryptophan residues in the protein are oxidized.
Of the conventional techniques listed above, quantitative proteome analysis using a stable isotope-labeled compound of sulfenyl halide does not suggest quantification of oxidized tryptophan residues.
An object of the present invention is to provide a method capable of determining the degree of oxidation of tryptophan residues in proteins and peptides, and a method capable of determining the position of oxidized tryptophan residues. .
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) preparing a protein sample A in which the degree of oxidation of tryptophan residues is unknown and a protein sample B in which the degree of oxidation of tryptophan residues is unknown;
(Ii) preparing two kinds of compounds having different molecular weights by containing isotopes having the same molecular structure and different mass numbers as the modifying reagent,
The protein sample A is modified with tryptophan residues using either one of the two compounds to obtain a modified protein sample A ′,
Separately, the protein sample B is modified with a tryptophan residue using the other of the two compounds to obtain a modified protein sample B ′,
(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; How to do.
In the above (1), the term “protein” is used to include a peptide having a relatively small molecular weight.
In (1) above, two types of protein samples are prepared in step (i), and the two types of protein samples are separately modified in step (ii) using two types of modifying reagents that differ only in the isotopic composition. (In other words, one of the protein samples is isotopically 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).
Further, 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 provided for the method of the present invention. That is, in the step (iii), a process selected from desalting, drying, resolubilization, reductive alkylation, fragmentation, concentration / separation, and fractionation as well as mixing is appropriately performed by those skilled in the art. May be done.
(2) The method for quantifying an oxidized tryptophan residue in a protein according to (1), wherein the modifying reagent is a sulfenyl compound.
(3) The method for quantifying an oxidized tryptophan residue in a protein according to (2), wherein the sulfenyl compound is an arylsulfenyl halide.
(4) The method for quantifying an oxidized tryptophan residue in a protein according to (3), wherein the arylsulfenyl halide is 2-nitrobenzenesulfenyl chloride.
The invention of the following (5) is a method for determining the oxidation position of a tryptophan residue of a protein.
(5) A method for determining the position of an oxidized tryptophan residue by performing the method according to any one of (1) to (4) and then determining the amino acid sequence.
The following inventions (6) to (9) are methods for quantifying the absolute amount of the degree of oxidation of a protein.
(6) (i) preparing a protein sample C for which the degree of oxidation of tryptophan residues is to be quantified and a protein sample D for which the degree of oxidation of tryptophan residues is known;
(Ii) preparing two kinds of compounds having different molecular weights by containing isotopes having the same molecular structure and different mass numbers as the modifying reagent,
The protein sample C is modified with a tryptophan residue using either one of the two compounds to obtain a modified protein sample C ′,
Separately, the protein sample D is modified with a tryptophan residue using 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) A method for quantifying oxidized tryptophan residues in a protein, comprising quantifying the degree of oxidation of tryptophan residues in the protein sample C by subjecting the mixed sample for mass spectrometry to mass spectrometry.
In the above (6), the term “protein” is used to include a peptide having a relatively small molecular weight.
In (6) above, two types of protein samples are prepared in step (i), and the two types of protein samples are separately modified in step (ii) using two types of modifying reagents that differ only in the isotopic composition. (In other words, one of the protein samples is isotopically 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 (6), the mixed sample for mass spectrometry obtained in 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 (iii), a process selected from desalting, drying, resolubilization, reductive alkylation, fragmentation, concentration / separation, and fractionation as well as mixing is appropriately performed by those skilled in the art. May be done.
(7) The method for quantifying an oxidized tryptophan residue in a protein according to (6), wherein the modifying reagent is a sulfenyl compound.
(8) The method for quantifying an oxidized tryptophan residue in a protein according to (7), wherein the sulfenyl compound is an arylsulfenyl halide.
(9) The method for quantifying an oxidized tryptophan residue in a protein according to (8), wherein the arylsulfenyl halide is 2-nitrobenzenesulfenyl chloride.
The invention of (10) below is a method for determining the oxidation position of a tryptophan residue of a protein.
(10) A method for determining the position of an oxidized tryptophan residue by performing the method according to any one of (6) to (9) and then determining the amino acid sequence.

FIG. 1 is a diagram outlining an example when protein samples A and B in which the degree of oxidation of tryptophan residues is unknown in the present invention are analyzed.
FIG. 2 shows a protein sample C in which the degree of oxidation of tryptophan residues is unknown and a non-oxidized protein in which the tryptophan residues are not oxidized (that is, the degree of oxidation is 0%) in the present invention. It is a figure outlining an example at the time of making sample D into analysis object.
FIG. 3 is an MS spectrum showing the results of quantification of the tryptophan residue oxidation degree of the present invention for a DSIP sample in which 50% of the tryptophan residue is oxidized using a tryptophan residue non-oxidized DSIP sample.

なお、タンパク質・ペプチド中のトリプトファン残基に対する修飾試薬としてのスルフェニル化合物については、国際公報第２００４／００２９５０パンフレットなどに詳述されている。
上記一般式２で表される化合物としては、ベンジルハライドが好ましい。さらに、ベンジルハライドとしては、２−ヒドロキシ−５−ニトロベンジルブロミド（２−ｈｙｄｒｏｘｙ−５−ｎｉｔｒｏｂｅｎｚｙｌ ｂｒｏｍｉｄｅ）などが挙げられる。
２−ヒドロキシ−５−ニトロベンジルブロミドを用いる場合、下記式に示すように、重い試薬としての２−ヒドロキシ−５−ニトロ［^１３Ｃ_６］ベンジルブロミドと、軽い試薬としての２−ヒドロキシ−５−ニトロ［^１２Ｃ_６］ベンジルブロミドとを組み合わせて用いることが好ましい。

なお、タンパク質・ペプチド中のトリプトファン残基に対する修飾試薬としてのベンジルハライド化合物については、Ｈｏｒｔｏｎ，Ｈ．Ｒ．ａｎｄ Ｋｏｓｈｌａｎｄ，Ｄ．Ｅ，Ｊｒ．（１９７２），Ｍｏｄｉｆｉｃａｔｉｏｎ ｏｆ ｐｒｏｔｅｉｎｓ ｗｉｔｈ ａｃｔｉｖｅ ｂｅｎｚｙｌ ｈａｌｉｄｅｓ，Ｍｅｔｈｏｄｓ ｉｎ ＥＮＺＹＭＯＬＯＧＹ，２５，４６８−４８２などに詳述されている。
修飾試薬としては、上に挙げた化合物に限らず、トリプトファン残基に対する修飾能を有し、且つ酸化トリプトファン残基に対する反応性を有さない化合物が、重い試薬と軽い試薬と組み合わされていれば特に限定されない。
（ｉｉｉ：質量分析用混合試料の調製）
上記の修飾工程によって得られた２種の修飾タンパク質試料Ａ’とＢ’、或いは２種の修飾タンパク質試料Ｃ’とＤ’とを互いに混合する。
質量分析用混合試料においては、混合された修飾タンパク質試料は、脱塩処理、乾燥処理、再可溶化処理、還元アルキル化処理、断片化処理、及び／又は、トリプトファン残基を有する分子について濃縮分離処理されたものであっても良い。
これらの処理を行う段階は、修飾タンパク質試料同士の混合段階の前後を問わず、当業者が適宜決定することができる。
これら処理を行うための方法は、いずれも限定されない。例えば脱塩工程は、セファデックスカラムなど、通常用いられる脱塩カラムを用いることによって行うことができる。再可溶化処理は、上述の可溶化処理と同様に行うことができる。還元アルキル化処理も、通常の方法によって行うことができる。断片化処理も特に限定されず、通常行われるトリプシンなどの酵素による消化や化学的断片化を行うことができる。
濃縮分離処理も特に限定されない。例えば、セファデックスカラムや、フェニル基を有する担体を充填したカラムを用いることができる。フェニル基を有する担体を充填したカラムは、上記（ｉｉ）工程における修飾試薬として、π電子性化合物（ＮＢＳＣｌ試薬を含む）が用いられる場合などに有用に用いられる。このようなカラムとしては、Ｈｉ−Ｔｒａｐ ｐｈｅｎｙｌ ＦＦ、Ｈｉ−Ｔｒａｐ ｐｈｅｎｙｌ ＨＰ、Ｐｈｅｎｙｌ Ｓｅｐｈａｒｏｓｅ ６ Ｆａｓｔ Ｆｌｏｗ、Ｐｈｅｎｙｌ Ｓｅｐｈａｒｏｓｅ Ｈｉｇｈ Ｐｅｒｆｏｒｍａｎｃｅ、（以上、アマシャムバイオサイエンス社製）、ＹＭＣ＊ＧＥＬ Ｐｈ（ワイエムシィ社製）などが挙げられる。
さらに、質量分析用混合試料は、分画処理が行われたものであっても良い。分画処理を行うための方法としては、逆相カラムとＨＰＬＣとを用いたシステムを用いる方法などが挙げられる。分画処理を行われた場合の質量分析用混合試料は、それぞれ、複数の画分を含むことになる。
（ｉｖ：質量分析）
上記質量分析用混合試料について、質量分析を行う。本発明における測定にはＭＡＬＤＩ型質量分析装置を用いることができる。この場合、ＭＡＬＤＩ−ＴＯＦ型質量分析装置（例えば島津製作所／クレイトス製ＡＸＩＭＡ−ＣＦＲ ｐｌｕｓ）等や、ＭＡＩＤＩ−ＩＴ−ＴＯＦ型質量分析装置（例えば島津製作所／クレイトス製ＡＸＩＭＡ−ＱＩＴ）等が用いられる。
ＭＡＬＤＩ質量分析装置を用いる場合、マトリックスとしては特に限定されない。例えば、ＤＨＢ（２，５−ジヒドロキシ安息香酸；２，５−ｄｉｈｙｄｒｏｘｙｂｅｎｚｏｉｃ ａｃｉｄ）、４−ＣＨＣＡ（α−シアノ−４−ヒドロキシ桂皮酸；α−ｃｙａｎｏ−４−ｈｙｄｒｏｘｙｃｉｎｎａｍｉｃ ａｃｉｄ）、３−ＣＨＣＡ（α−シアノ−３−ヒドロキシ桂皮酸；α−ｃｙａｎｏ−３−ｈｙｄｒｏｘｙｃｉｎｎａｍｉｃ ａｃｉｄ）、３Ｈ４ＮＢＡ（３−ヒドロキシ−４−ニトロ安息香酸；３−ｈｙｄｒｏｘｙ−４−ｎｉｔｒｏｂｅｎｚｏｉｃ ａｃｉｄ）などをマトリックスとして用いることができる。ＭＡＬＤＩ−ＴＯＦ型質量分析装置を用いる場合は、４−ＣＨＣＡをマトリックスとして用いると良い。一方、ＭＡＩＤＩ−ＩＴ−ＴＯＦ型質量分析装置を用いる場合は、３−ＣＨＣＡ、３Ｈ４ＮＢＡ、３Ｈ４ＮＢＡと４−ＣＨＣＡとの混合物をマトリックスとして用いると良い。
このような条件のもと、上記質量分析用混合試料について、質量分析装置による測定を行い、ＭＳスペクトルを得る。ＭＳスペクトル解析は、以下のようにして行う。
たとえば、上記工程（ｉ）で同量の２種のタンパク質試料を用意し、^１３Ｃ ＮＢＳＣｌ及び^１２Ｃ ＮＢＳＣｌを用いた上記工程（ｉｉ）と、断片化を含む上記工程（ｉｉｉ）とを行って質量分析用混合試料を得た場合を仮定する。上記工程（ｉ）で用意した２種のタンパク質試料のうち、少なくともいずれか一方のタンパク質試料において、トリプトファン残基が部分的に酸化されていた場合、すなわち、２種のタンパク質試料のうち、少なくともいずれか一方の試料中に、酸化トリプトファン残基を有するタンパク質（以下、ｏｘｉ−Ｔｒｙ含有タンパク質と記載する場合がある）が、非酸化トリプトファン残基を有するタンパク質（以下、ｎｏｎ−ｏｘｉ−Ｔｒｙ含有タンパク質と記載する場合がある）とともに含まれていた場合、得られる質量分析用混合試料には、ＮＢＳ修飾トリプトファン残基を有するペプチド（以下、ＮＢＳ−Ｔｒｙ含有ペプチドと記載することがある）と、酸化トリプトファン残基を有するペプチド（以下、ｏｘｉ−Ｔｒｙ含有ペプチドと記載することがある）とが少なくとも含まれる。ＮＢＳ−Ｔｒｙ含有ペプチドとしては、重い試薬で修飾された^１３ＣＮＢＳ−Ｔｒｙ含有ペプチドと軽い試薬で修飾された^１２ＣＮＢＳ−Ｔｒｙ含有ペプチドとが含まれる。
ＭＳスペクトルの解析においては、上記工程（ｉｉ）で用いた重い試薬と軽い試薬との分子量差に相当する質量電荷比を有するペアピーク（すなわちＮＢＳ−Ｔｒｙ含有ペプチドのピーク）に着目する。
ＮＢＳ試薬による修飾は、酸化されていないトリプトファン残基に対して起こることから、ペアピークを構成する２本のピークそれぞれの強度比は、上記（ｉ）で用意した２種のタンパク質試料中にそれぞれに含まれていたｎｏｎ−ｏｘｉ−Ｔｒｙ含有タンパク質の量の比に相当する。従って、ペアピークの強度の逆数比が、ｏｘｉ−Ｔｒｙ含有タンパク質の量の比に相当することになる。
このように、工程（ｉｖ）のＭＳ解析では、ペアピークを構成する２本のピークそれぞれの強度比から、タンパク質試料中の酸化度を求める。
また、工程（ｉ）で用意したタンパク質試料の量比と、工程（ｉｖ）で得られたＭＳスペクトルにおけるペアピークの強度比とが同じになる場合も考えられる。タンパク質試料の双方の酸化数が未知の場合にこのようなスペクトルが得られる場合、工程（ｉ）における２種のタンパク質試料のいずれにもｏｘｉ−Ｔｒｙ含有タンパク質が含まれていなかった可能性と、２種のタンパク質試料が同じ割合でｏｘｉ−Ｔｒｙ含有タンパク質を含んでいた可能性との両方が考えられる。そのため、工程（ｉｖ）のＭＳ解析では、ＮＢＳ−Ｔｒｙ含有ペプチドに相当する当該ペアピークとともに、ＮＢＳ−Ｔｒｙ含有ペプチドに対応する構造を有するｏｘｉ−Ｔｒｙ含有ペプチドに相当するシングルピークが検出されていることを確認することが好ましい（ここで、「ＮＢＳ−Ｔｒｙ含有ペプチドに対応する構造を有するｏｘｉ−Ｔｒｙ含有ペプチド」とは、トリプトファン残基がＮＢＳ化されているか酸化されているかの違いを除いてはＮＢＳ−Ｔｒｙ含有ペプチドと同じ構造を有するペプチドをいう）。このようなシングルピークが検出されていれば、２種のタンパク質試料が同じ割合でｏｘｉ−Ｔｒｙ含有タンパク質を含んでいたと判断することができる。
以下に、参考例をモデルに、本発明をより具体的に説明する。
［参考例１］
参考例１では、トリプトファン残基の酸化度が共に未知である、タンパク質試料Ａとタンパク質試料Ｂとを解析対象とした場合の一例を示す（図１参照）。
まず、タンパク質試料Ａ及びタンパク質試料Ｂを、同じ量だけ用意する（ｉ）。タンパク質試料Ａを、ＮＢＳＣｌ ｈｅａｖｙ試薬で^１３Ｃ ＮＢＳ修飾し、^１３Ｃ ＮＢＳ修飾タンパク質試料Ａ’を得る。一方で、タンパク質試料Ｂを、ＮＢＳＣｌ ｌｉｇｈｔ試薬で^１２Ｃ ＮＢＳ修飾し、^１２Ｃ ＮＢＳ修飾タンパク質試料Ｂ’を得る（ｉｉ）。^１３Ｃ ＮＢＳ修飾タンパク質試料Ａ’と^１２Ｃ ＮＢＳ修飾タンパク質試料Ｂ’とを混合／脱塩し、還元／アルキル化、トリプシン消化、及び濃縮を行い、質量分析用混合試料を調製する（ｉｉｉ）。得られた質量分析用混合試料を、質量分析装置を用いて測定する（ｉｖ）。得られるＭＳスペクトルにおいて、ＮＢＳＣｌ ｈｅａｖｙ試薬とＮＢＳＣｌ ｌｉｇｈｔ試薬との分子量差に相当する６Ｄａの質量差を有するペアピーク、すなわちＮＢＳ−Ｔｒｙ含有ペプチドのピークに着目する。図１の（ｉｖ）に、ＭＳスペクトル（横軸：質量電荷比（Ｍａｓｓ／Ｃｈａｒｇｅ）、縦軸：相対強度（％ Ｉｎｔ．））のモデルを示す。図１のように、当該ペアピークのうち、タンパク質試料Ａに由来する^１３Ｃ ＮＢＳ−Ｔｒｙ含有ペプチドのピーク強度と、タンパク質試料Ｂに由来する^１２Ｃ ＮＢＳ−Ｔｒｙ含有ペプチドのピーク強度との比が、３：７であったとする。タンパク質試料Ａとタンパク質試料Ｂとが同量用いられたことから、タンパク質試料Ａとタンパク質試料Ｂとにおける、ｏｘｉ−Ｔｒｙ含有タンパク質の量比は、７：３であったことが分かる。すなわち、参考例１においては、タンパク質試料Ａとタンパク質試料Ｂとのトリプトファン残基酸化度の相対比は、７：３であったことが分かる。
［参考例２］
参考例２では、トリプトファン残基の酸化度が未知であるタンパク質試料Ｃと、トリプトファン残基が酸化されていないこと（すなわち酸化度が０％であること）が分かっている非酸化タンパク質試料Ｄとを解析対象とした場合の一例を示す（図２参照）。
まず、タンパク質試料Ｃ及び非酸化タンパク質試料Ｄを、同じ量だけ用意する（ｉ）。タンパク質試料Ｃを、ＮＢＳＣｌ ｈｅａｖｙ試薬で^１３Ｃ ＮＢＳ修飾し、^１３Ｃ ＮＢＳ修飾タンパク質試料Ａ’を得る。一方で、非酸化タンパク質試料Ｄを、ＮＢＳＣｌ ｌｉｇｈｔ試薬で^１２Ｃ ＮＢＳ修飾し、^１２Ｃ ＮＢＳ修飾タンパク質試料Ｂ’を得る（ｉｉ）。その後は、上記参考例１と同様の工程（ｉｉｉ）及び（ｉｖ）を行う。得られたＭＳスペクトルにおいて、ペアピークに着目し、非酸化タンパク質試料Ｄに由来する^１２Ｃ ＮＢＳ−Ｔｒｙ含有ペプチドのピーク強度に対する、タンパク質試料Ｃに由来する^１３Ｃ ＮＢＳ−Ｔｒｙ含有ペプチドのピーク強度を調べる。図２のように、非酸化タンパク質試料Ｄに由来するピークの強度を１とした場合に、タンパク質試料Ｃに由来するピークの強度が０．２であったとする。タンパク質試料Ｃと非酸化タンパク質試料Ｄとが同量用いられたことから、参考例２においては、タンパク質試料Ｃのトリプトファン残基酸化度は８０％であったことが分かる。
本発明においてターゲットとしているトリプトファン残基は、タンパク質中での出現頻度が最も少ない部類に属するアミノ酸である。その頻度は、たとえば上記の工程（ｉｉｉ）で断片化処理を行った場合に得られるトリプトファン残基含有ペプチドフラグメントなどの、比較的鎖長の短いペプチド１分子につき、トリプトファン残基１個であることが多い。
この場合、本発明では、ＭＳ解析により酸化度が明らかになったタンパク質試料に関し、酸化されたトリプトファン残基の位置の決定を行うこともできる。具体的には、ＭＳ測定で得られたペアピークの少なくともいずれか一方をプレカーサイオンとしてＭＳ／ＭＳ測定、或いはそれ以上の多段階ＭＳ測定を行い、アミノ酸配列を決定する。アミノ酸配列を決定することは、トリプトファン残基の位置を特定することでもある。
ＭＳで得られたペアピーク自体は、タンパク質試料中のｎｏｎ−ｏｘｉ−Ｔｒｙ含有タンパク質に由来するものであるが、ｎｏｎ−ｏｘｉ−Ｔｒｙ含有タンパク質中のトリプトファン残基の位置を知ることによって、当該ｎｏｎ−ｏｘｉ−Ｔｒｙ含有タンパク質に対応する構造を有するｏｘｉ−Ｔｒｙ含有タンパク質（すなわち、トリプトファン残基が酸化されているか否かの違いを除いてはｎｏｎ−ｏｘｉ−Ｔｒｙ含有タンパク質と同じ構造を有するタンパク質）における酸化位置を間接的に知ることができる。(I: Preparation of protein sample)
First, two types of protein samples are prepared for examining the oxidation state of tryptophan residues. Examples of the two kinds of protein samples include those having the following relationship.
For example, when one protein is a protein sample derived from one sample and the other protein is a protein sample derived from another sample; one protein is a protein sample to be analyzed, and the other protein sample is When it is a control protein for one protein; when one protein sample is a protein sample extracted from a disease state 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 kinds of proteins used in the present invention so as to facilitate analysis in later mass spectrometry. At least one of the two proteins has an unknown degree of oxidation. If the degree of oxidation of the two proteins is different, the average molecular weights of the proteins in the two protein samples are strictly different. However, since the tryptophan residue targeted in the present invention is an amino acid belonging to a class with the lowest appearance frequency in proteins, the difference in the average molecular weight can be considered to be essentially negligible.
When the degree of oxidation of tryptophan residues in both of the two protein samples is unknown (in this case, the two protein samples may be referred to as protein samples A and B), the present invention The relative oxidation degree of the sample can be known. 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) The absolute degree of oxidation of the one protein sample can be known.
The protein sample may have been solubilized. The solubilization method is not particularly limited. For example, the 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 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. 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.
(Ii) Modification of protein sample In this step, one of two kinds of proteins is isotope-labeled by a so-called isotope labeling method. At this time, an unoxidized tryptophan residue is modified in both of the two types of proteins by the modifying reagent for performing the isotope labeling method.
As the modifying reagent, a compound that has the ability to selectively modify tryptophan residues and does not react with oxidized tryptophan residues can be used without particular limitation.
Tryptophan 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 tryptophan residue used in the present invention, when the tryptophan residue is not oxidized, it has reactivity to the 2-position of the side chain indole ring, and when the tryptophan residue is oxidized. A compound having such a characteristic that does not have reactivity is used.
In addition, the modifying reagents in the present invention are used in combination of two compounds having the same molecular structure but having different molecular weights by containing isotopes having different mass numbers. Among these, a compound having a large molecular weight is a heavy reagent, and a reagent having a small molecular weight is a light reagent. More specifically, a compound in which at least one element constituting the modifying reagent molecule is labeled with a stable isotope has the same structure, but the aforementioned element is a stable isotope different from the stable isotope. Used in combination with an isotope-labeled compound. Then, the compound labeled with a stable isotope having a larger 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 modified with one of a heavy reagent and a light reagent to obtain a modified protein sample A ′, and the protein sample B is mixed with a heavy reagent and The modified protein sample B ′ is obtained by modifying with one of the light reagents. 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 mixed with a heavy reagent and a light reagent. One of them is modified to obtain a modified protein sample D ′.
Examples of the modifying reagent that can be used in the present invention include compounds having a structure represented by the general formula 1: R ₁ —S—X ₁ (R ₁ represents an organic group, and X ₁ represents a leaving group). And a compound having a structure represented by the general formula 2: R ₂ -X ₂ (R ₂ represents an aralkyl group which may be substituted, and X ₂ represents a leaving group).
As the compound represented by the above general formula 1, that is, the sulfenyl compound, arylsulfenyl halide, in which R ₁ in the above general formula 1 is an aryl group which may be substituted, is preferable. Further, as the arylsulfenyl halide, 2-nitrobenzenesulfenyl chloride (NBSCl) is preferable.
When 2-nitrobenzenesulfenyl chloride is used, as shown in the following formula, 2-nitro [ ¹³ C ₆ ] benzenesulfenyl chloride (NBSCl heavy) as a heavy reagent and 2-nitro [ ¹² C ₆ as a light reagent are used. It is preferable to use in combination with benzenesulfenyl chloride (NBSCl light).
Both the NBSCl heavy reagent and the NBSCl light reagent are housed and sold in ¹³ C NBS ^(R) Stable Isotopic Labeling Kit-N manufactured by Shimadzu Corporation.

The sulfenyl compound as a modifying reagent for tryptophan residues in proteins and peptides is described in detail in International Publication No. 2004/002950 pamphlet and the like.
As the compound represented by Formula 2, benzyl halide is preferable. Furthermore, examples of the benzyl halide include 2-hydroxy-5-nitrobenzyl bromide.
When 2-hydroxy-5-nitrobenzyl bromide is used, 2-hydroxy-5-nitro [ ¹³ C ₆ ] benzyl bromide as a heavy reagent and 2-hydroxy-5-5 as a light reagent as shown in the following formula: It is preferable to use in combination with nitro [ ¹² C ₆ ] benzyl bromide.

The benzyl halide compound as a modifying reagent for tryptophan residues in proteins and peptides is described in Horton, H. et al. R. and Koshland, D.A. E, Jr. (1972), Modification of protein with active benzyl halides, Methods in ENZYMOLOGY, 25, 468-482.
The modifying reagent is not limited to the compounds listed above, but a compound that has the ability to modify tryptophan residues and does not have reactivity to oxidized tryptophan 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 modification step, or two kinds of modified protein samples C ′ and D ′ are mixed with each other.
In the mixed sample for mass spectrometry, the mixed modified protein sample is concentrated and separated with respect to molecules having desalting, drying, resolubilization, reductive alkylation, fragmentation, and / or tryptophan residues. It may be processed.
The stage of performing these treatments can be appropriately determined by those skilled in the art regardless of whether the modified protein sample is mixed or not.
Any method for performing these processes is not 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. The fragmentation treatment is also not particularly limited, and digestion or chemical fragmentation with an enzyme such as trypsin that 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 π-electron compound (including an NBSCl 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, etc. (manufactured by Amersham Bioscience, Inc., YMCG) Is mentioned.
Furthermore, the mixed sample for mass spectrometry may have been subjected to fractionation. Examples of the method for performing fractionation include a method using a system using a reverse phase column and HPLC. Each of the mixed samples for mass spectrometry when the fractionation process is performed includes a plurality of fractions.
(Iv: mass spectrometry)
Mass spectrometry is performed on the mixed sample for mass spectrometry. A MALDI mass spectrometer can be used for the measurement in the present invention. In this case, a MALDI-TOF type mass spectrometer (for example, AXIMA-CFR plus manufactured by Shimadzu Corporation / Kraitos) or a MAIDI-IT-TOF type mass spectrometer (for example, Shimadzu Corporation / Kraitos AXIMA-QIT) is used.
When using a MALDI mass spectrometer, the matrix is not particularly limited. For example, DHB (2,5-dihydroxybenzoic acid; 2,5-dihydroxybenzoic acid), 4-CHCA (α-cyano-4-hydroxycinnamic acid; α-cyan-4-hydroxycinnamic acid), 3-CHCA (α- Cyano-3-hydroxycinnamic acid; α-cyano-3-hydroxycinnamic acid), 3H4NBA (3-hydroxy-4-nitrobenzoic acid; 3-hydroxy-4-nitrobenzoic acid), and the like can be used as a matrix. When a MALDI-TOF mass spectrometer is used, 4-CHCA is preferably used as a matrix. On the other hand, when a MAIDI-IT-TOF type mass spectrometer is used, a mixture of 3-CHCA, 3H4NBA, 3H4NBA and 4-CHCA is preferably used as a matrix.
Under such conditions, the mass spectrometric sample is measured with respect to the mass spectrometric mixed sample 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 step (i), and the step (ii) using ¹³ C NBSCl and ¹² C NBSCl and the step (iii) including fragmentation are performed. Assume that a mixed sample for mass spectrometry is obtained. When tryptophan residues are partially oxidized in at least one of the two protein samples prepared in the step (i), that is, at least one of the two protein samples. In either sample, a protein having an oxidized tryptophan residue (hereinafter sometimes referred to as an oxi-Try-containing protein) is a protein having a non-oxidized tryptophan residue (hereinafter referred to as a non-oxy-Try-containing protein). In the mixed sample for mass spectrometry obtained, a peptide having an NBS-modified tryptophan residue (hereinafter sometimes referred to as an NBS-Try-containing peptide), and oxidized tryptophan are included. Peptides having residues (hereinafter referred to as oxi-Try-containing peptides) It is) and is included at least the mounting. NBS-Try-containing peptides include ¹³ CNBS-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 a pair peak having a mass-to-charge ratio corresponding to the molecular weight difference between the heavy reagent and the light reagent used in the step (ii) (that is, the peak of the NBS-Try-containing peptide).
Since the modification with the NBS reagent occurs on the non-oxidized tryptophan residue, the intensity ratio of each of the two peaks constituting the pair peak is in each of the two protein samples prepared in (i) above. This corresponds to the ratio of the amount of non-oxy-Try-containing protein contained. Therefore, the reciprocal ratio of the intensity of the paired peaks corresponds to the ratio of the amount of oxi-Try-containing protein.
Thus, in the MS analysis of the step (iv), 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 pair peak intensity ratio in the MS spectrum obtained in step (iv). When such a spectrum is obtained when the oxidation numbers of both of the protein samples are unknown, there is a possibility that neither of the two protein samples in step (i) contained the oxi-Try-containing protein. It is conceivable that both protein samples may have contained oxi-Try-containing proteins in the same proportion. Therefore, in the MS analysis of step (iv), a single peak corresponding to the oxi-Try-containing peptide having a structure corresponding to the NBS-Try-containing peptide is detected together with the pair peak corresponding to the NBS-Try-containing peptide. (Here, “an oxy-Try-containing peptide having a structure corresponding to an NBS-Try-containing peptide” means that except for the difference in whether the tryptophan residue is NBS or oxidized) Refers to a peptide having the same structure as an NBS-Try-containing peptide). If such a single peak is detected, it can be determined that the two protein samples contained the oxi-Try-containing protein at the same rate.
Hereinafter, the present invention will be described more specifically with reference examples.
[Reference Example 1]
Reference Example 1 shows an example of the case where protein sample A and protein sample B, in which both the degrees of oxidation of tryptophan residues are unknown, are analyzed (see FIG. 1).
First, the same amount of protein sample A and protein sample B is prepared (i). Protein sample A is ¹³ C NBS modified with NBSCl heavy reagent to obtain ¹³ C NBS modified protein sample A ′. On the other hand, protein sample B is ¹² C NBS modified with NBSCl light reagent to obtain ¹² C NBS modified protein sample B ′ (ii). ^{The 13} C NBS modified protein sample A ′ and the ¹² C NBS modified protein sample B ′ are mixed / desalted, reduced / alkylated, trypsin 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 spectrum, attention is paid to a pair peak having a mass difference of 6 Da corresponding to the molecular weight difference between the NBSCl heavy reagent and the NBSCl light reagent, that is, the peak of the NBS-Try-containing peptide. FIG. 1 (iv) shows a model of MS spectrum (horizontal axis: mass to charge ratio (Mass / Charge), vertical axis: relative intensity (% Int.)). As shown in FIG. 1, the ratio of the peak intensity of the ¹³ C NBS-Try-containing peptide derived from the protein sample A to the peak intensity of the ¹² C NBS-Try-containing peptide derived from the protein sample B among the paired peaks is Suppose that it was 3: 7. Since the same amount of the protein sample A and the protein sample B was used, it can be understood that the amount ratio of the oxi-Try-containing protein in 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 tryptophan residue oxidation between protein sample A and protein sample B was 7: 3.
[Reference Example 2]
In Reference Example 2, a protein sample C in which the degree of oxidation of tryptophan residues is unknown, and a non-oxidized protein sample D in which it is known that the tryptophan residues are not oxidized (that is, the degree of oxidation is 0%) An example of the case where is set as an analysis target is shown (see FIG. 2).
First, the same amount of protein sample C and non-oxidized protein sample D are prepared (i). Protein sample C is ¹³ C NBS modified with NBSCl heavy reagent to obtain ¹³ C NBS modified protein sample A ′. On the other hand, non-oxidized protein sample D is ¹² C NBS modified with NBSCl light reagent to obtain ¹² C NBS-modified protein sample B ′ (ii). Thereafter, the same steps (iii) and (iv) as in Reference Example 1 are performed. In the obtained MS spectrum, paying attention to the pair peak, the peak intensity of the ¹³ C NBS-Try-containing peptide derived from the protein sample C with respect to the peak intensity of the ¹² C NBS-Try-containing peptide derived from the non-oxidized protein sample D is examined. . 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 tryptophan residue oxidation degree of the protein sample C was 80%.
The tryptophan residue targeted in the present invention is an amino acid belonging to a class having the lowest occurrence frequency in proteins. The frequency is, for example, one tryptophan residue per peptide molecule having a relatively short chain length, such as a tryptophan residue-containing peptide fragment obtained when fragmentation is performed in the above step (iii). There are many.
In this case, in the present invention, the position of the oxidized tryptophan 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 multistage MS measurement using at least one of the pair peaks obtained by MS measurement as a precursor ion. Determining the amino acid sequence is also specifying the position of the tryptophan residue.
The pair peak itself obtained by MS is derived from the non-oxy-Try-containing protein in the protein sample, but by knowing the position of the tryptophan residue in the non-oxy-Try-containing protein, In an oxi-Try-containing protein having a structure corresponding to the oxi-Try-containing protein (that is, a protein having the same structure as the non-oxy-Try-containing protein except for whether or not tryptophan residues are oxidized) The oxidation position can be known indirectly.

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 (DSIP: WAGGDASGE (SEQ ID NO: 1), m / z 848.1) was used. Prepare a DSIP model sample in which 50% tryptophan residues are oxidized (that is, a sample containing equal amounts of oxidized DSIP and non-oxidized DSIP), and determine the degree of oxidation of the model sample using 100% non-oxidized DSIP as a control. went.
1. Preparation of DSIP oxidant 1) Preparation of formic acid 900 μL of formic acid and 100 μL of hydrogen peroxide (30 w / w%) were mixed and allowed to react on ice for 2 hours.
2) Oxidation of DSIP 100 μg of DSIP (manufactured by Peptide Institute) was dissolved in 200 μL of a 0.1 v / v% trifluoroacetic acid (TFA) aqueous solution. To the obtained DSIP solution, 100 μL of formic acid 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 DSIP model sample in which 50% of tryptophan residues are oxidized The total amount of the DSIP oxidant prepared in (1) was dissolved in 50 μL of acetic acid to obtain a DSIP oxidant solution. Separately, 100 μg of non-oxidized DSIP was dissolved in 50 μL of a 0.1 v / v% TFA aqueous solution to obtain a non-oxidized DSIP solution. The non-oxidized DSIP solution was added to the DSIP oxidant solution and mixed. The obtained mixture corresponds to a sample containing 200 μg of DSIP in which 50% of tryptophan residues are oxidized in a mixed solution of acetic acid-0.1 v / v% TFA aqueous solution (volume ratio 1: 1).
3. Determination of degree of oxidation of tryptophan residue in model sample by NBS reagent ( ¹³ C NBS ^(R) Stable Isotope Labeling Kit-N, manufactured by Shimadzu Corporation) Non-oxidized DSIP (200 μg) was added to acetic acid-0.1 v / v% TFA aqueous solution. (Volume ratio 11) Dissolved in a mixed solution to obtain a non-oxidized DSIP sample. To a non-oxidized DSIP sample, a solution of ¹² C NBS reagent (1 mg) in acetic acid (100 μL) was added. On the other hand, the above 2. A solution of ¹³ C NBS reagent (1 mg) in acetic acid (100 μL) was added to the DSIP model sample prepared in (1). Both samples were allowed to react for 3 hours at room temperature. After completion of the reaction, 2 μL aliquots were taken from both samples, mixed with each other, and further diluted with 200 μL of 0.1 v / v% TFA aqueous solution. After performing the ZipTip treatment, MALDI-TOF MS was measured with a mass spectrometer (AXIMA-CFR, manufactured by Shimadzu Corporation) using α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix.
4). Results of MALDI-TOF MS On the MS spectrum, DSIP pair peaks modified with the NBS reagent were observed at m / z 1002.58 and 1008.64. The area ratio of each peak was Area (1002.58): Area (1008.64) = 1.99: 1.
This is a sample in which a peak having a larger mass-to-charge ratio is modified with ¹³ C NBS reagent, that is, a DSIP model sample having oxidized tryptophan residues, and the degree of oxidation of tryptophan residues in the model sample is 50%. It shows that there is. This result was in good agreement with the DSIP model sample containing 50% DSIP in which tryptophan residues were oxidized and 50% DSIP in which tryptophan residues were not oxidized. From this, it was shown that the degree of tryptophan residue oxidation 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 was obtained from the intensity of the pair peak even if the oxidation degree of at least one of the two types of samples was unknown.
In the above Examples, it was confirmed that the effect of the present invention was achieved by artificially preparing a protein sample having a tryptophan residue oxidation degree of 50% and analyzing this sample. Therefore, the present invention can naturally be carried out on a protein sample having a natural degree of tryptophan residue oxidation. In the above examples, the analysis was performed using the DSIP sample as the protein sample to be analyzed and the NBS reagent as the modifying reagent for performing the isotope labeling method. However, the present invention also applies to protein samples other than DSIP samples and isotope labeling modifying reagents other than NBS reagents. Therefore, the said Example is only a mere illustration in all points, and must not be interpreted limitedly. Further, all modifications belonging to the equivalent scope of the claims are within the scope of the present invention.
[Sequence Listing]

Claims (10)

(I) preparing a protein sample A in which the degree of oxidation of tryptophan residues is unknown and a protein sample B in which the degree of oxidation of tryptophan residues is unknown;
(Ii) preparing two kinds of compounds having different molecular weights by containing isotopes having the same molecular structure and different mass numbers as the modifying reagent,
The protein sample A is modified with tryptophan residues using either one of the two compounds to obtain a modified protein sample A ′,
Separately, the protein sample B is modified with a tryptophan residue using the other of the two compounds to obtain a modified protein sample B ′,
(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; How to do. The method for quantifying oxidized tryptophan residues in a protein according to claim 1, wherein the modifying reagent is a sulfenyl compound. The method for quantifying an oxidized tryptophan residue in a protein according to claim 2, wherein the sulfenyl compound is an arylsulfenyl halide. The method for quantifying an oxidized tryptophan residue in a protein according to claim 3, wherein the arylsulfenyl halide is 2-nitrobenzenesulfenyl chloride. A method for determining the position of an oxidized tryptophan residue in a protein by performing the method according to any one of claims 1 to 4 and then performing amino acid sequencing. (I) preparing a protein sample C in which the degree of oxidation of tryptophan residues is to be quantified and a protein sample D in which the degree of oxidation of tryptophan residues is known;
(Ii) preparing two kinds of compounds having different molecular weights by containing isotopes having the same molecular structure and different mass numbers as the modifying reagent,
The protein sample C is modified with a tryptophan residue using either one of the two compounds to obtain a modified protein sample C ′,
Separately, the protein sample D is modified with a tryptophan residue using 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) A method for quantifying oxidized tryptophan residues in a protein, comprising quantifying the degree of oxidation of tryptophan residues in the protein sample C by subjecting the mixed sample for mass spectrometry to mass spectrometry. The method for quantifying an oxidized tryptophan residue in a protein according to claim 6, wherein the modifying reagent is a sulfenyl compound. The method for quantifying an oxidized tryptophan residue in a protein according to claim 7, wherein the sulfenyl compound is an arylsulfenyl halide. The method for quantifying an oxidized tryptophan residue in a protein according to claim 8, wherein the arylsulfenyl halide is 2-nitrobenzenesulfenyl chloride. A method for determining the position of an oxidized tryptophan residue in a protein by performing the method according to any one of claims 6 to 9 and then performing amino acid sequencing.

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