JP4524823B2 - Method for determining amino acid sequence of amino terminal of protein - Google Patents

Description

【００２７】
実施例２
リジンを含有し、かつ、Ｎ末端が修飾されていないペプチドとしてダイノルフィンＡ（アミノ酸配列；YGGFLRRIRPKLKWDNQ、ペプチド研究所製）100pmolを0.1Mピリジン-酢酸（pH6.0）、0.1Mピリジン-酢酸（pH5.0）および0.1%酢酸水溶液（pH3.3）各12μLにそれぞれ溶解し、氷上で１分間インキュベートした後、0.1Mイソシアン酸フェニル／無水テトラヒドロフランを5μL添加し、氷上で５分間インキュベートした。該反応液から溶媒を減圧下に留去し、残渣を質量分析用試料とした。
質量分析は二重収束型質量分析計（日本電子、型式JMS-HX/HX110A）を用い、FABイオン化法を使用した。上記試料を水・メタノール・酢酸（５０・５０・１）2μLに溶解し、該試料溶液1μLをグリセロール・チオグリセロール（１・１）1μLと混合して上記分析計に供し、FAB-MSおよびFAB-MS/MS測定を行った。
その結果、使用した全ての溶媒において原料のダイノルフィンＡに比し質量が119増加したピークが検出された。MS/MS分析を行ったところ、該ピークがＮ末端α-アミノ基のみが修飾されたダイノルフィンＡであることが確認できた。
【００２８】
実施例３
(1) Ｎ末端が修飾されたタンパク質であるウマチトクロムｃ（シグマ社製）200μgを1%炭酸水素アンモニウム溶液200μLに溶解し、1mg/mLキモトリプシン4μLを添加し、37℃で18時間インキュベートした。
(2) 前記キモトリプシン消化物の反応液10μLを減圧濃縮し、残渣に0.1Mピリジン-酢酸（pH 5.0）を24μL添加し、半量をアセチル化用検体として以下の処理を行い、半量をアセチル化しないコントロール用検体として分取した。アセチル化検体は、氷上で１分間インキュベートした後、0.1M無水酢酸／無水テトラヒドロフランを5μL添加し、氷上で５分間インキュベートした。反応後、溶媒を減圧下に留去し、残渣を質量分析用反応試料とした。別途前記コントロール用検体の溶媒を減圧下に留去し、残渣を質量分析用コントロール試料とした。
質量分析は実施例１と同じ装置を使用し、各試料を水・メタノール・酢酸（５０・５０・１）2μLに溶解し、試料溶液１μLをグリセロール・チオグリセロール（１・１）１μLと混合して質量分析計に供し、FAB-MS測定を行った。
図１にアセチル化処理前後の試料のFAB-MSスペクトルを示す。コントロール試料において検出されたウマチトクロムｃ由来のピークのうちm/z1162.5のピークはアセチル化処理後の試料においても検出されたが、それ以外のピークはアセチル化処理後の試料において検出されず、それぞれ質量が42増加したピークが検出された。従って、m/z1162.5のピークがウマチトクロムｃのＮ末端由来のペプチドと特定できた。このm/z1162.5ピークのFAB-MS/MSスペクトルを取得し、Ｎ末端構造をAc-GDVEKGKKIFと決定した。尚、このＮ末端構造は既に知られているチトクロムcのＮ末端構造と一致していた。
【００２９】
実施例４
実施例３の(1)で得られたキモトリプシン消化物の反応液2μLを減圧濃縮し、残渣に0.1Mピリジン-酢酸（pH 6.0）を24μL添加し、半量をイソシアン酸フェニル処理用検体として以下の処理を行い、半量をイソシアン酸フェニルで処理しないコントロール用検体として分取した。
前記イソシアン酸フェニル処理用検体を氷上で１分間インキュベートした後、これに0.1Mイソシアン酸フェニル／無水テトラヒドロフランを5μL添加し、氷上で５分間インキュベートした。反応後、溶媒を減圧下に留去し、残渣を質量分析用反応試料とした。別途前記コントロール用検体の溶媒を減圧下に留去し、残渣を質量分析用コントロール試料とした。
質量分析は実施例１と同じ装置を使用し、試料を水・メタノール・酢酸（５０・５０・１）2μLに溶解し、試料溶液１μLをグリセロール・チオグリセロール（１・１）１μLと混合して質量分析計に供し、FAB-MS測定した。
図２にイソシアン酸フェニル処理前後の試料のFAB-MSスペクトルを示す。コントロール試料において検出されたウマチトクロムｃ由来のピークのうちm/z1162.5のピークはイソシアン酸フェニル処理後の試料においても検出されたが、それ以外のピークはイソシアン酸フェニル処理後の試料において検出されず、それぞれ質量が119増加したピークが検出された。従って、m/z1162.5のピークがウマチトクロムｃのＮ末端由来のペプチドと同定できた。
【００３０】
実施例５
(1) Ｎ末端が修飾されていないタンパク質である組換えヒト成長ホルモン（以下、hGHと記す。）200pmolを0.1Mピリジン-酢酸（pH 5.0）12μLに溶解し、氷上で１分間インキュベートした後、0.1M無水酢酸／無水テトラヒドロフランを5μL添加し、氷上で５分間インキュベートした。反応後、溶媒を減圧下に留去した。
(2) (1)で得られたＮ末端選択的アセチル化処理したhGH200pmolを100mM炭酸水素アンモニウム溶液50μLに溶解し、0.1mg/mLトリプシン5μLを添加し、37℃で18時間インキュベートした。前記トリプシン消化物の反応液を減圧濃縮し、反応試料を調製した。
(3)(1)で得られたＮ末端選択的アセチル化処理したhGHに代えて、(1)の原料として用いたhGHを用いた以外は(2)と同様の操作を行い、コントロール試料を調製した。
(4) 質量分析は二重収束型質量分析計（日本電子、型式JMS-HX/HX110A）を用い、FABイオン化法を使用した。反応試料、コントロール試料のそれぞれを水・メタノール・酢酸（５０・５０・１）4μLに溶解し、試料溶液1μLをグリセロール・チオグリセロール（１・１）1μLと混合して質量分析計に供し、FAB-MS測定した。
図３にアセチル化処理前後の試料（処理前：コントロール試料、処理後：反応試料）のFAB-MSスペクトルを示す。コントロール試料において検出されたhGH由来のピークのうちm/z930.4のピークは反応試料において質量が42増加したピークとなって検出された。従って、m/z930.4のピークがhGHのＮ末端由来のペプチドと特定できた。このm/z930.4ピークのFAB-MS/MSスペクトルを取得し、Ｎ末端構造をFPTIPLSRと決定した。尚、該構造は既に知られているhGHのＮ末端構造と一致した。
【００３１】
実施例６
(1) Ｎ末端が修飾されていないタンパク質である組換えヒト成長ホルモン（以下、hGHと記す。）を試料として使用して電気泳動ゲル上のタンパク質のＮ末端の解析を行った。電気泳動はMultiphorIIシステム（ファルマシア）、SDS-PAGE用ゲルExcelGel SDS gradient 8-18（ファルマシア）を用い、hGH試料0.5μgを泳動した。泳動後のゲルはクーマシーブリリアントブルー（以下、CBBと記す。）を水／メタノール／酢酸（４０・６０・１）で0.1%濃度に調製した溶液で10分間浸して染色し、水／メタノール／酢酸（４０・６０・１）中で１時間浸して脱色を行った。CBB染色操作後、hGHに相当するバンドを切り出した。
(2) システインの還元アルキル化処理を以下のように行った。ゲルを1.5mLのチューブに入れ、50μLの0.1M炭酸水素アンモニウム溶液と50μLのアセトニトリルを添加して３０分浸透した。この操作を３回繰り返した後、10mMジチオスレイトール／0.1M炭酸水素アンモニウム溶液を添加して１時間56℃で保温した。溶液を廃棄した後、55mMヨードアセトアミド／0.1M炭酸水素アンモニウムを添加して室温で45分反応させた。溶液を廃棄した後、50μLの0.1M炭酸水素アンモニウム溶液と50μLのアセトニトリルを添加して３０分浸透する操作を３回繰り返し、ゲルに含まれている溶媒を減圧下で留去した。
(3) 前項により得られた二組の還元アルキル化処理したゲルを準備し、一つはイソシアン酸フェニルで処理し、もう一つはイソシアン酸フェニルで処理しないコントロールゲルとした。還元アルキル化処理したゲルに12μLの0.1Mピリジン−酢酸（pH6.0）と5μLの0.1M イソシアン酸フェニル／無水テトラヒドロフランを添加し、氷中で10分反応させた後、減圧下で溶媒を留去することでフェニルイソシアネート処理した。
(4) (3)で得られたイソシアン酸フェニル処理したゲルおよびコントロールゲルに0.1mg/mLのトリプシンを5μLと15μLの0.1M炭酸水素アンモニウムを添加して、37℃で18時間反応させることによってタンパク質を切断した。切断後、ゲルに水／アセトニトリル／ぎ酸（５０・５０・５）を50μL添加して40分間浸透させ、上清を集める操作を２回繰り返して、切断ペプチドを抽出し、溶媒を減圧下に留去した後、残渣を質量分析に供した。
質量分析は二重収束型質量分析計（日本電子、型式JMS-HX/HX110A）を用い、FABイオン化法を使用した。グリセロール・チオグリセロール（１・１）を水・メタノール・酢酸（５０・５０・１）で２０％に希釈したマトリックスを使用した。試料を水・メタノール・酢酸（５０・５０・１）1μLに溶解し、試料溶液１μLをマトリックス１μLと混合して質量分析計に供し、FAB-MS測定した。コントロール試料において検出されたhGH由来のピークのうちm/z930.4のピークはイソシアン酸フェニル処理後の試料において質量が119増加したピークが検出された。従って、m/z930.4のピークがhGHのＮ末端由来のペプチドと特定できた。このm/z930.4ピークのFAB-MS/MSスペクトルを取得し、Ｎ末端構造をFPTIPLSRと決定した。尚、この構造は既に知られているhGHのＮ末端構造と一致した。
【００３２】
実施例７
実施例1において無水酢酸に代えて表２に記載のイソシアネート化合物〔２〕を用い、表２に記載した条件とした以外は実施例１と同様に実験を行った。結果を表２に示す。
【００３３】
【表２】

＊１：シアネート化合物の添加量を添加したシアネート化合物／ＴＨＦ５μＬ中のシアネート化合物のモル濃度（ｍＭ）として表示する。
＊２：Ｎ末端のみアセチル化されたダイノルフィンＡの選択率
◎：７５％超
○：７０％超７５％以下
△：６０％超７０％以下
×：６０％以下
【００３４】
【発明の効果】
本発明によれば、タンパク質のＮ末端アミノ酸配列を効率的に決定する方法を提供できる。また、タンパク質やペプチドのＮ末端アミノ基を効率的に修飾することが可能となる。
【図面の簡単な説明】
【図１】ウマチトクロムcのキモトリプシン消化物について、Ｎ末端α-アミノ基選択的アセチル化処理前後の試料の質量スペクトルを対比して示す。上段はアセチル化処理前の試料の質量スペクトルであり、図中の●印をつけたピークがウマチトクロムcのキモトリプシン消化物に由来する。下段はアセチル化処理後の試料の質量スペクトルである。
【図２】ウマチトクロムcのキモトリプシン消化物について、Ｎ末端α-アミノ基選択的なイソシアン酸フェニル処理前後の試料の質量スペクトルを対比して示す。上段はイソシアン酸フェニル処理前の試料の質量スペクトルであり、図中の●印をつけたピークがウマチトクロムcのキモトリプシン消化物に由来する。下段はイソシアン酸フェニル処理後の試料の質量スペクトルである。
【図３】Ｎ末端α-アミノ基選択的アセチル化処理前後のhGHのトリプシン消化物について、質量スペクトルを対比して示す。上段はアセチル化処理前の試料の質量スペクトルであり、図中の●印をつけたピークがhGHのトリプシン消化物に由来する。下段はアセチル化処理後の試料の質量スペクトルである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for determining the amino terminal amino acid sequence of a protein and a method for selectively modifying the amino terminal amino group of a protein or peptide.
[0002]
[Prior art]
Determination of the amino acid sequence of a protein is important as a means for confirming and identifying the structure of the protein. In particular, determining the amino acid sequence of the amino terminal portion of a protein is important for obtaining genes encoding useful proteins.
There are two types of naturally occurring proteins, one with a modified amino terminus (hereinafter referred to as the N terminus) and the other with no modification. For example, Edman degradation method is used for N-terminal amino acid sequencing of a protein whose N-terminal is not modified (hereinafter referred to as N-terminal unmodified protein), that is, a protein having no substituent at the N-terminal amino group. Has been. On the other hand, Edman degradation is used to determine the N-terminal amino acid sequence of N-terminal modified proteins (hereinafter referred to as N-terminal modified proteins), that is, proteins in which the hydrogen atom of the terminal amino group is substituted with an acetyl group or the like. Since this is not possible, mass spectrometry or the like is used for that purpose. For example, the amino acid sequence of each peptide can be determined by cleaving the protein with a digestive enzyme or the like and subjecting the resulting peptides to tandem mass spectrometry.
[0003]
In order to determine the N-terminal amino acid sequence of the N-terminal modified protein, it is necessary to identify the peptide derived from the N-terminal of the original protein from the peptides generated by cleaving the protein and determine the amino acid sequence of this peptide There is. As a method for identifying a peptide derived from the N-terminus of an N-terminal modified protein, a peptide generated by cleaving the protein (hereinafter referred to as a cleaved peptide) is treated with a BrCN-activated Sepharose column, and the peptide eluted without being adsorbed is obtained. By collecting the sample (Anal. Biochem., 222, 210-216 (1994)) or by acetylating the cleaved peptide and comparing the elution positions in the reverse phase chromatography of the sample before and after the acetylation treatment, A method for identifying a peptide derived from the N-terminus of a protein (Japanese Patent Laid-Open No. 8-27180) is known. These all have an ε-amino group derived from a lysine residue in addition to the cleaved peptide N-terminal α-amino group. Since the ε-amino group derived from this lysine residue also reacts in the same manner as the reagent etc., in order to specify the peptide derived from the N-terminal of the protein, the ε-amino group of the protein lysine residue is succinylated, for example. It was necessary to modify the N-terminal α-amino group of the cleaved peptide and specify the protein after cleaving it in advance.
[0004]
On the other hand, as a selective modification method of N-terminal α-amino group of protein or cleaved peptide, acetylation method using acetic anhydride in glucagon (Eur. J. Biochem. 60, 335-347, (1975)), cleavage Although a method of using iodoacetic anhydride as a coupling reagent for peptides (Anal. Chem., 65, 1703, (1993); Bioconjugate Chem., 1, 114-122, (1990)) is known, For the selectivity in the method, it was necessary to strictly control the use ratio of the protein or peptide as a substrate and the modifying reagent.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for efficiently determining the amino acid sequence of the N-terminal portion of a protein and a method for selectively modifying the N-terminal amino group of a protein, peptide or the like.
[0006]
[Means for Solving the Problems]
Under such circumstances, the present inventors have studied a method for determining the N-terminal amino acid sequence of a protein, and as a result, the ε-- within a relatively wide range without strictly controlling the amount of the modifier used for the protein or cleaved peptide. The present inventors have found a method capable of modifying an N-terminal amino group with extremely high selectivity even in the presence of an amino group, and found that the object can be efficiently achieved by applying this method to N-terminal amino acid sequencing of a protein. The present invention has been reached.
That is, the present invention
(1) cleaving a protein having a modified amino terminus by chemical or biochemical means;
(2) The peptide obtained by the process of (1) has a general formula [1] at pH 5 or lower.
(R ¹ CO) ₂ O [1]
(In the formula, R ¹ represents an alkyl group.)
Or a general formula [2] at pH 6 or lower
R ² NCO [2]
(Wherein R ² represents an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group, or an optionally substituted pyridyl group.)
A step of allowing an isocyanate compound represented by
(3) performing a mass analysis of the peptide before and after the step of (2), comparing both, and specifying a peptide derived from the amino terminus of the protein; and
(4) a method for determining the amino-terminal amino acid sequence of a protein having a modified amino terminus, comprising the step of determining the amino acid sequence of the identified peptide;
(a) a protein having an amino terminal not modified at a pH of 5 or less and represented by the general formula [1]
(R ¹ CO) ₂ O [1]
(In the formula, R ¹ represents an alkyl group.)
Or a general formula [2] at pH 6 or lower
R ² NCO [2]
(Wherein R ² represents an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group, or an optionally substituted pyridyl group.)
A step of allowing an isocyanate compound represented by
(b) a step of cleaving each protein before and after the step of (a) by chemical or biochemical means,
(c) performing mass spectrometry of the peptides obtained in the steps of (b), comparing the two and identifying peptides derived from the amino terminus of the protein, and
(d) The present invention relates to a method for determining the amino terminal amino acid sequence of a protein whose amino terminal is not modified, which comprises the step of determining the amino acid sequence of the identified peptide.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
In the present invention, protein refers to a polymer compound composed of polypeptide chains in which α-amino acids are linked by peptide bonds, and usually refers to a molecular weight of 4000 or more. A peptide refers to a peptide in which two or more α-amino acids are linked by peptide bonds, and generally refers to a peptide having 50 or fewer amino acids. The term “modified at the amino terminus” means that at least one hydrogen atom of the amino group at the amino terminus of the protein or peptide is substituted with an acyl group or the like.
[0009]
1. Method for determining amino acid sequence of amino terminal of N-terminal modified protein
(1) N-terminal modified protein cleavage step The cleavage method of the N-terminal modified protein is not particularly limited. For example, A Practical Guide to Protein and Peptide Purification for Microsequencing, Second Edition, Academic Press, (1993) and Methods in Enzymology, 193, 389-412, (1990) and the like, biochemical cleavage methods using digestive enzymes, and the like. Specifically, for example, the chemical cleavage method includes a cleavage method using BrCN, and the biochemical cleavage method using a digestive enzyme includes digestion using trypsin, chymotrypsin, or the like.
By cleaving the N-terminal modified protein, a peptide having a molecular weight of 3000 or less, usually cleaved from the protein, is obtained.
In addition, when the N-terminal modified protein contains cysteine as an amino acid constituting it, it is practical to cleave the protein after performing a reduction S alkylation treatment with, for example, iodoacetamide or 4-vinylpyridine in advance. Above preferred. This reduction S alkylation process itself is known and can be performed according to the method described in, for example, A Practical Guide to Protein and Peptide Purification for Microsequencing, Second Edition, Academic Press, (1993).
[0010]
(2) Treatment with carboxylic acid anhydride or isocyanate compound to peptide i) Carboxylic acid anhydride [1] treatment The peptide obtained in the above step is reacted with carboxylic acid anhydride [1] at a pH of 5 or less to reduce the N-terminus. A selectively acylated peptide is obtained.
R ¹ in the carboxylic acid anhydride [1] is a linear or branched alkyl group, preferably a C1-6 linear or branched alkyl group such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, or a hexyl group. Specific examples include acetic anhydride, propionic anhydride, butyric anhydride, valeric anhydride, and the like.
[0011]
The reaction in this step is carried out at a pH of 5 or less, preferably in the range of pH 2 to 5, more preferably in the range of pH 3 to 5.
In this step, a buffer solution for maintaining the pH is usually used as a solvent. As the solvent, volatile solvents such as an acetic acid aqueous solution, a formic acid aqueous solution, a pyridine-acetic acid buffer solution, and a trifluoroacetic acid aqueous solution are preferable from the viewpoint of ease of post-treatment. Usually, the reaction in this step is performed by adding a solution such as carboxylic acid anhydride [1] or tetrahydrofuran thereof to a solution or suspension composed of the peptide and the solvent. The reaction is preferably carried out at a temperature in the range of about 10 ° C. or lower and the solvent used does not freeze from the viewpoint of selectivity. The reaction time is usually about 1 to 60 minutes.
The concentration of carboxylic acid anhydride [1] in the reaction system is usually about 10 to 30 mM when the reaction is carried out at pH 5 as the initial concentration in the reaction system, and about 3 if the reaction is carried out at pH 3.3. It is about 60-300 mM.
After the reaction in this step, the peptide is recovered from the reaction solution. For example, when a volatile solvent is used for the reaction solution, the reaction solution may be distilled off under reduced pressure. When a solvent containing a non-volatile salt is used, after the desalting treatment, the solvent may be distilled off under reduced pressure.
By carrying out the treatment in this step, it is possible to produce an acylated peptide corresponding to the carboxylic acid anhydride [1] selectively used only at the N-terminus.
[0012]
ii) Isocyanate compound [2] treatment The peptide obtained by cleaving the N-terminal modified protein is allowed to react with the isocyanate compound [2] at pH 6 or lower to obtain a peptide in which the N-terminal amino group is selectively carbamoylated. .
R ² in the isocyanate compound [2] is an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group, or an optionally substituted pyridyl group. Examples of the alkyl group which may be substituted include an alkyl group which may be substituted with a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom and an iodine atom. Specifically, a methyl group , C1-6 alkyl groups such as ethyl group, propyl group, butyl group, pentyl group, hexyl group, and C1-6 haloalkyl groups such as chloromethyl group, iodomethyl group, dibromomethyl group, trifluoromethyl group, iodoethyl group, etc. be able to. Examples of the optionally substituted phenyl group and optionally substituted naphthyl group include halogen-substituted phenyl groups such as phenyl group, p-chlorophenyl group, o-bromophenyl group, 2,4-dichlorophenyl group, 1- A naphthyl group such as a naphthyl group and a 2-naphthyl group, a halogen-substituted naphthyl group such as a 1-iodonaphthyl group and a 2-chloronaphthyl group, a phenyl group having a fluorescent substituent such as a fluorescein group and a dansylaminophenyl group Can be mentioned. Examples of the optionally substituted pyridyl group include pyridyl groups such as 2-pyridyl group, 3-pyridyl group, and 4-pyridyl group, 2-chloro-4-pyridyl group, and 3,5-difluoro-2-pyridyl. And halogen-substituted pyridyl groups such as a group.
Specific examples of the isocyanate compound [2] include phenyl isocyanate, p-chlorophenyl isocyanate, 1-naphthyl isocyanate, 2-naphthyl isocyanate, butyl isocyanate, ethyl isocyanate, 4-pyridyl isocyanate, 3-pyridyl isocyanate, fluorescein isocyanate, Examples thereof include dansylaminophenyl isocyanate.
The reaction in this step is performed at a pH of 6 or less, preferably in the range of about pH 3 to 6, more preferably about pH 5-6.
[0013]
In this step, a buffer solution for maintaining the pH is usually used as a solvent. As the solvent, volatile solvents such as an acetic acid aqueous solution, a formic acid aqueous solution, a pyridine-acetic acid buffer solution, and a trifluoroacetic acid aqueous solution are preferable from the viewpoint of ease of post-treatment. Usually, the reaction in this step is performed by adding a solution such as an isocyanate compound [2] or tetrahydrofuran thereof to a solution or suspension composed of the peptide and the solvent. Examples of the reaction include a range from a low temperature where the solvent used does not freeze to about 50 ° C. The reaction time is usually about 1 to 60 minutes.
The concentration of the isocyanate compound [2] in the reaction system is usually about 2 to 300 mM when the reaction is carried out at pH 6 as the initial concentration in the reaction system.
After the reaction in this step, the peptide is recovered from the reaction solution. For example, when a volatile solvent is used for the reaction solution, the reaction solution may be distilled off under reduced pressure. When a solvent containing a non-volatile salt is used, after the desalting treatment, the solvent may be distilled off under reduced pressure.
By carrying out the treatment in this step, it is possible to produce a peptide in which only the N-terminus is selectively carbamoylated corresponding to the isocyanate compound [2] to be used.
[0014]
(3) Identification of peptides derived from the amino terminus of proteins
The mass analysis of the peptide before the step (2) and the mass analysis of the peptide after the step (2) are performed, and the peptide whose mass has not changed is specified. It can be said that it is a peptide derived from. For example, when the peptide is treated with acetic anhydride, the peptide containing the N-terminus of the N-terminal modified protein does not change in mass due to the treatment, so the same mass peak is detected before and after step (2). In contrast, in a peptide that does not include the N-terminus of the N-terminal modified protein, a mass increase of 42 is detected by acetylation of the α-amino group at the N-terminus of the peptide. In addition, for example, when treated with phenyl isocyanate, a mass increase of 119 is detected for peptides that do not include the N-terminus of the N-terminal modified protein.
[0015]
As a method of mass spectrometry, any method capable of analyzing the mass of a peptide may be used, and the type of mass spectrometer and the ionization method are not particularly limited. For example, it can be analyzed by the FAB ionization method using a double-focusing mass spectrometer.
(4) Step of determining the amino acid sequence of the identified peptide The peptide derived from the N-terminus of the protein thus identified can be determined, for example, by subjecting the peptide to tandem mass spectrometry. As a method for tandem mass spectrometry, a method applicable in the step (3) can be similarly used.
[0016]
2. Method for determining amino-terminal amino acid sequence of N-terminal unmodified protein
(a) Carboxylic anhydride or isocyanate compound treatment step to N-terminal unmodified protein i) Carboxylic acid anhydride [1] By allowing carboxylic acid anhydride [1] to act on N-terminal unmodified protein at pH 5 or lower A protein acylated corresponding to the carboxylic acid anhydride [1] selectively used at the N-terminus is obtained.
In the process of this step, the above 1. (2) In place of the peptide in step i), except that an N-terminal unmodified protein is used, (2) The method of step i) can be similarly applied.
[0017]
ii) Isocyanate compound [2] treated N-terminal unmodified protein is allowed to react with isocyanate compound [2] at a pH of 6 or less to produce a carbamoylated protein corresponding to isocyanate compound [2] selectively used at the N-terminus. can get.
In the process of this step, the above 1. (2) 1. An N-terminal unmodified protein is used instead of the peptide in step ii). (2) The method of step ii) can be applied in the same manner.
In addition, when the N-terminal unmodified protein contains cysteine as an amino acid constituting the protein, after carrying out a reduction S alkylation treatment with, for example, iodoacetamide or 4-vinylpyridine in advance, the carboxylic acid anhydride to the protein It is practically preferable to carry out the treatment [1] or the isocyanate compound [2]. This reduction S alkylation treatment can also be performed in the same manner as the treatment for the N-terminal modified protein.
[0018]
(b) Protein cleavage step before and after step (a)
By cleaving each of the N-terminal unmodified protein used as a raw material in the step (a) and the protein obtained in the step (a) by chemical or biochemical means, each protein is cleaved. In general, a peptide having a molecular weight of 3000 or less is obtained.
Regarding the method for cleaving the protein, Instead of the N-terminal modified protein used in the step (1), an N-terminal unmodified protein used as a raw material in the step (a) and a protein obtained in the step (a) are used. The method of the step (1) can be applied similarly.
[0019]
(c) Identifying peptide derived from amino terminus of protein
obtained in the step (b), mass spectrometry of the peptide obtained by cleaving the N-terminal unmodified protein used as a raw material in the step (a), and cleaving the protein obtained in the step (a) Then, mass analysis of the obtained peptide is performed, and by comparing the two and specifying a peptide in which a mass change is detected, this can be specified as a peptide derived from the N-terminus of the protein. For example, when a protein is subjected to N-terminal selective acetylation treatment as described above, there is no change in mass before and after treatment in a peptide that does not include the N-terminus of the protein, whereas a peptide that includes the N-terminus of the protein , A mass increase of 42 is detected by acetylation of the N-terminal α-amino group. Also, for example, when treated with phenyl isocyanate, 119 mass gain is detected for peptides containing the N-terminus of the protein.
The method for mass spectrometry of the peptide is not particularly limited as long as it is a method capable of analyzing the mass change of the peptide, and the type of the mass spectrometer and the ionization method are not particularly limited. For example, the mass change of the cleaved peptide can be detected by FAB ionization using a double-focusing mass spectrometer.
[0020]
(d) Step of determining the amino acid sequence of the specified peptide The peptide derived from the N-terminus of the protein thus specified can be determined by subjecting the peptide to, for example, tandem mass spectrometry. As a method for tandem mass spectrometry, a method applicable in the step (c) can be similarly used.
The amino-terminal amino acid sequence determination method of the N-terminal modified protein of the present invention and the amino-terminal amino acid sequence determination method of the N-terminal unmodified protein are in a state where the protein separated by electrophoresis is held in a gel. It is also possible to apply.
[0021]
The amino-terminal amino acid sequence determination method of the N-terminal modified protein of the present invention and the amino-terminal amino acid sequence determination method of the N-terminal unmodified protein are capable of parallel processing of multiple samples. It can be measured quickly by mass spectrometry. Furthermore, since these steps can be automated, multiple samples can be processed quickly.
[0022]
By the method described above, it becomes possible to quickly determine the N-terminal sequences of many proteins. For example, by comparing with the genomic sequence, it is possible to quickly determine the start codon and identify the translation initiation signal.
[0023]
Furthermore, by subjecting proteins in general to N-terminal α-amino group-selective modification treatment and analyzing the mass before and after the treatment by mass spectrometry, a protein whose mass change is not detected before and after the treatment is originally N It can also be determined that the terminal is a modified protein. For example, an appropriate method can be selected in advance for analyzing the N-terminal amino acid sequence.
In addition, for proteins in general, N-terminal α-amino group selective modification treatment is performed in the same manner as described above, and the proteins before and after the treatment are analyzed by isoelectric focusing or two-dimensional electrophoresis. It is also possible to determine that a protein in which no change in isoelectric point is detected is a protein whose N-terminus is originally modified.
[0024]
【Example】
Hereinafter, although a reference example and an example explain the present invention still in detail, the present invention is not limited to these.
Example 1
100 pmol of dynorphin A (amino acid sequence; YGGFLRRIRPKLKWDNQ, manufactured by Peptide Institute, Inc.) as a peptide containing lysine and not modified at the N-terminus is added with 0.1 M pyridine-acetic acid (pH 6.0), 0.1 M pyridine-acetic acid (pH 5) 0.0), 0.1% acetic acid aqueous solution (pH 3.3) and 1% acetic acid aqueous solution (pH 2.7), each dissolved in 12 μL, incubated on ice for 1 minute, and then 5 μL of 0.005 M to 5 M acetic anhydride / tetrahydrofuran Added and incubated on ice for 5 minutes. The solvent was distilled off from the reaction solution under reduced pressure, and the residue was used as a sample for mass spectrometry.
For mass spectrometry, a double-focusing mass spectrometer (JEOL, model JMS-HX / HX110A) was used, and the FAB ionization method was used. Dissolve the above sample in 2 μL of water / methanol / acetic acid (50/50/1), mix 1 μL of the sample solution with 1 μL of glycerol / thioglycerol (1.1), and use it for the above analyzer. FAB-MS and FAB -MS / MS measurement was performed.
When MS / MS analysis was performed on the peak having a mass increased by 42 compared to the raw material dynorphin A after acetic anhydride treatment, it was confirmed that only the N-terminal α-amino group was acetylated dynorphin A. .
Table 1 shows the selectivity of dynorphin A acetylated only at the N-terminus under each pH and acetic anhydride amount condition.
[0025]
Explanation of symbols in Table 1 Selectivity of dynorphin A acetylated only at the N-terminal A: over 75% B: over 70% over 75% Δ: over 60% over 70% x: up to 60%
[Table 1]

[0027]
Example 2
100 pmol of dynorphin A (amino acid sequence; YGGFLRRIRPKLKWDNQ, manufactured by Peptide Institute, Inc.) as a peptide containing lysine and not modified at the N-terminus is added with 0.1 M pyridine-acetic acid (pH 6.0), 0.1 M pyridine-acetic acid (pH 5) 0.0) and 0.1% acetic acid aqueous solution (pH 3.3), each dissolved in 12 μL, and incubated on ice for 1 minute. Then, 5 μL of 0.1 M phenyl isocyanate / anhydrous tetrahydrofuran was added and incubated on ice for 5 minutes. The solvent was distilled off from the reaction solution under reduced pressure, and the residue was used as a sample for mass spectrometry.
For mass spectrometry, a double-focusing mass spectrometer (JEOL, model JMS-HX / HX110A) was used, and the FAB ionization method was used. Dissolve the above sample in 2 μL of water / methanol / acetic acid (50/50/1), mix 1 μL of the sample solution with 1 μL of glycerol / thioglycerol (1.1), and use it for the above analyzer. FAB-MS and FAB -MS / MS measurement was performed.
As a result, a peak having a mass increase of 119 compared to the raw material dynorphin A was detected in all the solvents used. When MS / MS analysis was performed, it was confirmed that the peak was dynorphin A in which only the N-terminal α-amino group was modified.
[0028]
Example 3
(1) 200 μg of horse cytochrome c (manufactured by Sigma), which is a protein modified at the N-terminus, was dissolved in 200 μL of a 1% ammonium hydrogen carbonate solution, 4 μL of 1 mg / mL chymotrypsin was added, and incubated at 37 ° C. for 18 hours.
(2) Concentrate 10 µL of the chymotrypsin digested reaction solution under reduced pressure, add 24 µL of 0.1M pyridine-acetic acid (pH 5.0) to the residue, and perform the following treatment using half of the sample for acetylation. The sample was collected as a control sample. The acetylated specimen was incubated on ice for 1 minute, and then 5 μL of 0.1M acetic anhydride / tetrahydrofuran was added and incubated on ice for 5 minutes. After the reaction, the solvent was distilled off under reduced pressure, and the residue was used as a reaction sample for mass spectrometry. Separately, the solvent of the control sample was distilled off under reduced pressure, and the residue was used as a control sample for mass spectrometry.
For mass spectrometry, the same apparatus as in Example 1 was used. Each sample was dissolved in 2 μL of water, methanol, and acetic acid (50, 50, 1), and 1 μL of the sample solution was mixed with 1 μL of glycerol / thioglycerol (1.1). And subjected to FAB-MS measurement.
FIG. 1 shows FAB-MS spectra of the sample before and after acetylation treatment. Among the peaks derived from horse cytochrome c detected in the control sample, the peak at m / z 1162.5 was also detected in the sample after acetylation treatment, but the other peaks were not detected in the sample after acetylation treatment. Each peak increased by 42. Therefore, the peak at m / z 1162.5 could be identified as a peptide derived from the N-terminus of equine cytochrome c. A FAB-MS / MS spectrum of this m / z 1162.5 peak was obtained, and the N-terminal structure was determined to be Ac-GDVEKGKKIF. This N-terminal structure was consistent with the known N-terminal structure of cytochrome c.
[0029]
Example 4
2 μL of the reaction solution of the chymotrypsin digest obtained in (3) of Example 3 was concentrated under reduced pressure, 24 μL of 0.1M pyridine-acetic acid (pH 6.0) was added to the residue, and half of the sample was treated with phenyl isocyanate as follows. Treatment was performed, and half of the sample was collected as a control sample not treated with phenyl isocyanate.
The phenyl isocyanate-treated specimen was incubated for 1 minute on ice, and then 5 μL of 0.1 M phenyl isocyanate / anhydrous tetrahydrofuran was added thereto, and incubated for 5 minutes on ice. After the reaction, the solvent was distilled off under reduced pressure, and the residue was used as a reaction sample for mass spectrometry. Separately, the solvent of the control sample was distilled off under reduced pressure, and the residue was used as a control sample for mass spectrometry.
For mass spectrometry, the same apparatus as in Example 1 was used. The sample was dissolved in 2 μL of water, methanol, and acetic acid (50, 50, 1), and 1 μL of the sample solution was mixed with 1 μL of glycerol and thioglycerol (1.1). It used for the mass spectrometer and measured FAB-MS.
FIG. 2 shows FAB-MS spectra of the sample before and after the phenyl isocyanate treatment. Among the peaks derived from horse cytochrome c detected in the control sample, the peak at m / z 1162.5 was also detected in the sample after phenyl isocyanate treatment, but the other peaks were detected in the sample after phenyl isocyanate treatment. In each case, peaks with a mass increase of 119 were detected. Therefore, the peak at m / z 1162.5 could be identified as a peptide derived from the N-terminus of equine cytochrome c.
[0030]
Example 5
(1) 200 pmol of recombinant human growth hormone (hereinafter referred to as “hGH”), a protein whose N-terminal is not modified, is dissolved in 12 μL of 0.1M pyridine-acetic acid (pH 5.0), and incubated on ice for 1 minute. 5 μL of 0.1M acetic anhydride / tetrahydrofuran was added and incubated on ice for 5 minutes. After the reaction, the solvent was distilled off under reduced pressure.
(2) The N-terminal selective acetylated hGH200pmol obtained in (1) was dissolved in 50 mM of 100 mM ammonium bicarbonate solution, 5 mg of 0.1 mg / mL trypsin was added and incubated at 37 ° C. for 18 hours. The reaction solution of the trypsin digest was concentrated under reduced pressure to prepare a reaction sample.
(3) Perform the same operation as in (2) except that hGH used as a raw material in (1) was used instead of the N-terminal selective acetylated hGH obtained in (1). Prepared.
(4) Mass spectrometry was performed using a FAB ionization method using a double-focusing mass spectrometer (JEOL, model JMS-HX / HX110A). Each of the reaction sample and the control sample is dissolved in 4 μL of water, methanol, and acetic acid (50, 50, 1), 1 μL of the sample solution is mixed with 1 μL of glycerol and thioglycerol (1.1), and used for the mass spectrometer. -MS measurement.
FIG. 3 shows FAB-MS spectra of samples before and after acetylation treatment (before treatment: control sample, after treatment: reaction sample). Among the peaks derived from hGH detected in the control sample, the peak at m / z 930.4 was detected as a peak having an increased mass of 42 in the reaction sample. Therefore, the peak at m / z 930.4 could be identified as a peptide derived from the N-terminus of hGH. A FAB-MS / MS spectrum of this m / z 930.4 peak was obtained, and the N-terminal structure was determined to be FPTIPLSR. The structure coincided with the known N-terminal structure of hGH.
[0031]
Example 6
(1) Recombinant human growth hormone (hereinafter referred to as hGH), which is a protein whose N-terminus is not modified, was used as a sample to analyze the N-terminus of the protein on the electrophoresis gel. Electrophoresis was performed using a Multiphor II system (Pharmacia) and SDS-PAGE gel ExcelGel SDS gradient 8-18 (Pharmacia), and 0.5 μg of hGH sample was run. The gel after electrophoresis was stained by immersing Coomassie Brilliant Blue (hereinafter referred to as CBB) in water / methanol / acetic acid (40 · 60 · 1) to a concentration of 0.1% for 10 minutes, and water / methanol / Decolorization was performed by immersing in acetic acid (40, 60, 1) for 1 hour. After the CBB staining operation, a band corresponding to hGH was cut out.
(2) Reductive alkylation treatment of cysteine was performed as follows. The gel was placed in a 1.5 mL tube, 50 μL of 0.1 M ammonium hydrogen carbonate solution and 50 μL of acetonitrile were added and permeated for 30 minutes. After this operation was repeated three times, 10 mM dithiothreitol / 0.1 M ammonium hydrogen carbonate solution was added, and the mixture was kept at 56 ° C. for 1 hour. After discarding the solution, 55 mM iodoacetamide / 0.1 M ammonium hydrogen carbonate was added and reacted at room temperature for 45 minutes. After discarding the solution, the operation of adding 50 μL of 0.1 M ammonium bicarbonate solution and 50 μL of acetonitrile and infiltrating for 30 minutes was repeated three times, and the solvent contained in the gel was distilled off under reduced pressure.
(3) Two sets of reductive alkylation-treated gels obtained in the previous section were prepared. One was treated with phenyl isocyanate and the other was a control gel not treated with phenyl isocyanate. 12 μL of 0.1 M pyridine-acetic acid (pH 6.0) and 5 μL of 0.1 M phenyl isocyanate / anhydrous tetrahydrofuran were added to the gel subjected to reductive alkylation treatment, reacted for 10 minutes in ice, and then the solvent was distilled off under reduced pressure. By leaving, it was treated with phenyl isocyanate.
(4) By adding 5 μL of 0.1 mg / mL trypsin and 15 μL of 0.1 M ammonium hydrogen carbonate to the phenyl isocyanate-treated gel and control gel obtained in (3) and reacting at 37 ° C. for 18 hours. The protein was cleaved. After cleaving, 50 μL of water / acetonitrile / formic acid (50, 50, 5) is added to the gel and allowed to permeate for 40 minutes, and the supernatant is collected twice to extract the cleaved peptide and the solvent under reduced pressure. After evaporation, the residue was subjected to mass spectrometry.
For mass spectrometry, a double-focusing mass spectrometer (JEOL, model JMS-HX / HX110A) was used, and the FAB ionization method was used. A matrix obtained by diluting glycerol / thioglycerol (1.1) with water / methanol / acetic acid (50/50/1) to 20% was used. The sample was dissolved in 1 μL of water, methanol, and acetic acid (50, 50, 1), 1 μL of the sample solution was mixed with 1 μL of the matrix, and the mass spectrometer was used for FAB-MS measurement. Among the peaks derived from hGH detected in the control sample, the peak at m / z 930.4 was detected with a mass increased by 119 in the sample after phenyl isocyanate treatment. Therefore, the peak at m / z 930.4 could be identified as a peptide derived from the N-terminus of hGH. A FAB-MS / MS spectrum of this m / z 930.4 peak was obtained, and the N-terminal structure was determined to be FPTIPLSR. This structure was consistent with the known N-terminal structure of hGH.
[0032]
Example 7
The experiment was conducted in the same manner as in Example 1 except that the isocyanate compound [2] shown in Table 2 was used instead of acetic anhydride in Example 1 and the conditions shown in Table 2 were used. The results are shown in Table 2.
[0033]
[Table 2]

* 1: The amount of cyanate compound added is expressed as the molar concentration (mM) of the cyanate compound in 5 μL of the added cyanate compound / THF.
* 2: Selectivity of dynorphin A acetylated only at the N-terminal ◎: over 75% ○: over 70% over 75% Δ: over 60% over 70% x: up to 60%
【The invention's effect】
The present invention can provide a method for efficiently determining the N-terminal amino acid sequence of a protein. It also becomes possible to efficiently modify the N-terminal amino group of proteins and peptides.
[Brief description of the drawings]
FIG. 1 shows a comparison of mass spectra of samples before and after N-terminal α-amino group selective acetylation of a chymotrypsin digest of horse cytochrome c. The upper row is the mass spectrum of the sample before acetylation treatment, and the peak marked with ● in the figure is derived from a chymotrypsin digest of horse cytochrome c. The lower row is the mass spectrum of the sample after acetylation treatment.
FIG. 2 shows a comparison of mass spectra of samples before and after N-terminal α-amino group-selective phenyl isocyanate treatment for a chymotrypsin digest of horse cytochrome c. The upper row is the mass spectrum of the sample before the treatment with phenyl isocyanate, and the peak marked with ● is derived from the chymotrypsin digest of horse cytochrome c. The lower row is the mass spectrum of the sample after treatment with phenyl isocyanate.
FIG. 3 shows a comparison of mass spectra of tryptic digests of hGH before and after N-terminal α-amino group selective acetylation. The upper row is the mass spectrum of the sample before acetylation treatment, and the peak marked with ● in the figure is derived from the digested product of hGH trypsin. The lower row is the mass spectrum of the sample after acetylation treatment.

Claims (10)

(1) a step of cleaving a protein having a modified amino terminus by chemical or biochemical means, which contains a lysine residue and the ε-amino group of the lysine residue is not protected in advance ;
(2) The amino terminal α- amino group of the amino groups of the peptide obtained in the process of (1) is selectively reduced to pH 5 or less and represented by the general formula [1]
(R ¹ CO) ₂ O [1]
(In the formula, R ¹ represents an alkyl group.)
Or a general formula [2] at pH 6 or lower
R ² NCO [2]
(Wherein R ² represents an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group, or an optionally substituted pyridyl group.)
A step of allowing an isocyanate compound represented by
(3) Perform mass analysis of the peptide before and after the step of (2), compare the two to find a peak without mass change and a peak with increased mass, and identify a peak without mass change from the amino terminus of the protein. Identifying as a peptide-based peak; and
(4) A method for determining the amino terminal amino acid sequence of a protein having a modified amino terminal, comprising the step of determining the amino acid sequence of a peptide corresponding to the identified peak. (1) The amino terminal α- amino group of the amino groups of proteins whose amino terminal is not modified is selectively expressed by the general formula [1]
(R ¹ CO) ₂ O [1]
(In the formula, R ¹ represents an alkyl group.)
Or a general formula [2] at pH 6 or lower
R ² NCO [2]
(Wherein R ² represents an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group, or an optionally substituted pyridyl group.)
A step of allowing an isocyanate compound represented by
(2) a step of cleaving each protein before and after the step of (1) by chemical or biochemical means,
(3) Mass spectrometry of the peptide obtained in the process of (2) is performed, and both are compared to find a peak without mass change and a peak with increased mass. The peak without mass change is derived from the amino terminus of the protein. Identifying as a peptide-based peak of
(4) A method for determining the amino terminal amino acid sequence of a protein whose amino terminal is not modified, comprising a step of determining an amino acid sequence of a peptide corresponding to the identified peak. (1) a step of cleaving a protein having a modified amino terminus by chemical or biochemical means, which contains a lysine residue and the ε-amino group of the lysine residue is not protected in advance ;
(2) The amino terminal α- amino group of the amino groups of the peptide obtained in the process of (1) is selectively reduced to pH 5 or less and represented by the general formula [1]
(R ¹ CO) ₂ O [1]
(In the formula, R ¹ represents an alkyl group.)
Or a general formula [2] at pH 6 or lower
R ² NCO [2]
(Wherein R ² represents an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group, or an optionally substituted pyridyl group.)
A step of reacting an isocyanate compound represented by:
(3) Perform mass analysis of the peptide before and after the step of (2), compare the two to find a peak without mass change and a peak with increased mass, and identify a peak without mass change from the amino terminus of the protein. A method for identifying a peptide derived from the amino terminus of a protein having a modified amino terminus, comprising the step of identifying the peak corresponding to the peptide. (1) The amino terminal α- amino group of the amino groups of proteins whose amino terminal is not modified is selectively expressed by the general formula [1]
(R ¹ CO) ₂ O [1]
(In the formula, R ¹ represents an alkyl group.)
Or a general formula [2] at pH 6 or lower
R ² NCO [2]
(Wherein R ² represents an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group, or an optionally substituted pyridyl group.)
A step of allowing an isocyanate compound represented by
(2) a step of cleaving each of the proteins before and after the step of (1) by chemical or biochemical means, and
(3) Mass spectrometry of the peptide obtained in the process of (2) is performed, and both are compared to find a peak without mass change and a peak with increased mass. The peak without mass change is derived from the amino terminus of the protein. A method for identifying a peptide derived from the amino terminus of a protein whose amino terminus is not modified, comprising a step of identifying the peptide as a peak based on the peptide. Prior to the step (1), reductive S-alkylation is performed on a protein having a modified amino terminus, which contains a lysine residue and the ε-amino group of the lysine residue is not protected in advance. Item 4. The method according to Item 1 or 3. The method according to claim 2 or 4, wherein reductive S-alkylation is performed on a protein whose amino terminus is not modified prior to the step (1). The amino terminal α- amino group of the amino group of a protein or peptide, which contains a lysine residue and the ε-amino group of the lysine residue is not protected in advance, is generally selected at a pH of 5 or less. Formula [1]
(R ¹ CO) ₂ O [1]
(In the formula, R ¹ represents an alkyl group.)
A method for acylating an amino-terminal α-amino group of a protein or peptide, which comprises reacting the carboxylic acid anhydride represented by The amino terminal α- amino group of the amino group of a protein or peptide, which contains a lysine residue and the ε-amino group of the lysine residue is not protected in advance, is generally selected at a pH of 6 or less. Formula [2]
R ² NCO [2]
(Wherein R ² represents an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group, or an optionally substituted pyridyl group.)
A method for carbamoylation of an amino-terminal α-amino group of a protein or peptide, which comprises reacting the isocyanate compound represented by the formula: The method according to any one of claims 1 to 7, wherein R ^{1 of the} carboxylic acid anhydride [1] is a C 1-6 alkyl group. The method according to claim 1, wherein R ^{2 of the} isocyanate compound [2] is a phenyl group or a C 1-6 alkyl group.

Images