JPH06189784A - Fractionation of n-terminal fragment of peptide - Google Patents
Fractionation of n-terminal fragment of peptideInfo
- Publication number
- JPH06189784A JPH06189784A JP35949592A JP35949592A JPH06189784A JP H06189784 A JPH06189784 A JP H06189784A JP 35949592 A JP35949592 A JP 35949592A JP 35949592 A JP35949592 A JP 35949592A JP H06189784 A JPH06189784 A JP H06189784A
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- JP
- Japan
- Prior art keywords
- terminal
- peptide
- amino group
- fragment
- group
- Prior art date
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6818—Sequencing of polypeptides
- G01N33/6824—Sequencing of polypeptides involving N-terminal degradation, e.g. Edman degradation
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- Hematology (AREA)
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- Biomedical Technology (AREA)
- Biophysics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、タンパク質やペプチド
のアミノ末端（以下、Ｎ末端と略す）フラグメントを分
取することからなる、ペプチドＮ末端フラグメントの分
取方法に関する。TECHNICAL FIELD The present invention relates to a method for fractionating an N-terminal fragment of a peptide, which comprises fractionating an amino-terminal (hereinafter abbreviated as N-terminal) fragment of a protein or peptide.
【０００２】[0002]
【従来の技術・発明が解決しようとする課題】従来、ペ
プチドのＮ末端およびＣ末端のアミノ酸配列の分析を行
うこと等を目的とするペプチド末端のフラグメントの分
取において、メカニカルロスやコンタミネーションが起
きやすいこと、さらに、操作が煩雑であることや比較的
多量のサンプルが必要なこと、配列決定に不確実性が生
じる等の問題点を有している。これらの欠点を克服した
改良方法として、Ｃ末端ペプチドの分取方法としては、
ペプチドをＬｙｓ−Ｃ特異切断酵素により切断処理した
後、生じた各フラグメントのαアミノ基およびεアミノ
基と反応して共有結合を形成しうる官能基を有する固体
に結合させ、次いで各ペプチドのαアミノ末端アミノ酸
残基と隣接するアミノ酸残基との間のペプチド結合を酸
処理により切断し、遊離のＣ末端ペプチドを分取する方
法（特開平１−２３５６００号公報）が知られており、
さらに同公報には、この分取方法により得られたＣ末端
ペプチドを用いて各種アミノ酸配列分析法によりアミノ
酸配列分析が可能であると開示されている。2. Description of the Related Art Conventionally, in the fractionation of peptide-terminal fragments for the purpose of analyzing the N-terminal and C-terminal amino acid sequences of peptides, mechanical loss and contamination have been There are problems that it is easy to occur, that the operation is complicated, that a relatively large amount of sample is required, and that there is uncertainty in sequencing. As an improved method that overcomes these drawbacks, a method for fractionating the C-terminal peptide is as follows:
After the peptide is cleaved with a Lys-C specific cleaving enzyme, it is bound to a solid having a functional group capable of reacting with the α-amino group and the ε-amino group of each resulting fragment to form a covalent bond, and then the α of each peptide is bound. A method is known in which a peptide bond between an amino-terminal amino acid residue and an adjacent amino acid residue is cleaved by acid treatment to separate a free C-terminal peptide (JP-A-1-235600),
Further, the same publication discloses that the C-terminal peptide obtained by this fractionation method can be used for amino acid sequence analysis by various amino acid sequence analysis methods.
【０００３】しかしながら、Ｎ末端ペプチドの分取方法
においては、前記の問題点が未だ充分解決されていると
はいえない。すなわち、サンプルが微量である場合、Ｎ
末端ペプチドの分取が困難であり、分取後のアミノ酸の
配列分析を最後まで完全に行うことができないという欠
点やメカニカルロス、コンタミネーションの発生といっ
た問題点が解決されておらず、これらを解決したＮ末端
ペプチドの分取方法の開発および一連の操作が自動化さ
れた実用的なＮ末端ペプチド分取装置の開発が望まれて
いる。However, it cannot be said that the above-mentioned problems have been sufficiently solved in the method for fractionating N-terminal peptides. That is, if the sample is very small, N
It is difficult to collect the terminal peptide, and the problems such as the inability to completely analyze the amino acid sequence after the separation, the problems such as mechanical loss and the occurrence of contamination have not been solved. It is desired to develop a method for fractionating the above N-terminal peptide and a practical N-terminal peptide fractionating apparatus in which a series of operations are automated.
【０００４】[0004]
【課題を解決するための手段】前記問題点を解決し、微
量なサンプルであっても完全かつ効率よくペプチドの分
取を行うとともに、引き続きアミノ酸配列の分析を行う
ことが可能で、しかも簡便な方法であるペプチドＮ末端
フラグメントの分取方法について鋭意検討した結果、本
発明を完成するに至った。[Means for Solving the Problems] It is possible to solve the above-mentioned problems, to perform complete and efficient peptide fractionation even in a small amount of sample, and to perform subsequent amino acid sequence analysis. The present invention has been completed as a result of extensive studies on a method for collecting a peptide N-terminal fragment, which is a method.
【０００５】即ち、本発明は、アミノ末端（Ｎ末端）の
αアミノ基がブロックされたペプチドにおいて、Ｌｙｓ
のεアミノ基をアセチル化し、該アセチル化体を化学的
処理および／または酵素的処理を行い、得られた各フラ
グメントのαアミノ基およびＬｙｓのεアミノ基と反応
して共有結合を形成しうる官能基を有する固体と各フラ
グメントとを反応させ、次いで遊離のＮ末端フラグメン
トをアリルアミン型官能基で誘導体化されたポリマーの
膜またはガラス繊維濾紙上で分取し、分取されたＮ末端
フラグメントを水溶性カルボジイミド等を用いて該Ｎ末
端フラグメント中のカルボキシル基を介して、該ポリマ
ーの膜または該ガラス繊維濾紙上に共有結合により固定
化することを特徴とする、ペプチドＮ末端フラグメント
の分取方法に関する。That is, the present invention provides a peptide in which the α-amino group at the amino terminus (N terminus) is blocked, Lys
The ε-amino group of γ-amino acid can be acetylated, and the acetylated product can be chemically and / or enzymatically treated to react with the α-amino group of each obtained fragment and the ε-amino group of Lys to form a covalent bond. The functionalized solid is reacted with each fragment, and the free N-terminal fragment is then fractionated on a polymer membrane or glass fiber filter paper derivatized with an allylamine-type functional group to remove the fractionated N-terminal fragment. A method for fractionating a peptide N-terminal fragment, which comprises immobilizing it by covalent bond on the polymer membrane or the glass fiber filter paper through a carboxyl group in the N-terminal fragment using water-soluble carbodiimide or the like. Regarding
【０００６】以下に、本発明を具体的に説明する。本発
明の方法では、まず、Ｎ末端のαアミノ基がブロックさ
れた目的のペプチド（タンパク質）の、Ｌｙｓのεアミ
ノ基をアセチルイミダゾールまたは無水酢酸等でアセチ
ル化を行なう。ここで、無水酢酸を用いた場合、後述の
Ｎ2 パージによりほぼ完全に除去が可能である。The present invention will be specifically described below. In the method of the present invention, first, the ε-amino group of Lys of the target peptide (protein) in which the α-amino group at the N-terminal is blocked is acetylated with acetylimidazole or acetic anhydride. Here, when acetic anhydride is used, it can be almost completely removed by the N 2 purge described later.
【０００７】次に、化学的および／または酵素的処理を
行う。本発明において公知の化学的処理とは、化学的手
法によりペプチドのクリーベイジを行う処理をさす。該
処理として具体的には、例えばＣＮＢｒによるＭｅｔ残
基での切断や、ＣＮＢｒ−ＤＭＳＯ−ＨＣｌによるＴｒ
ｐ残基での切断等が挙げられるが、前記の作用を有する
ものであれば特に限定されるものではない。本発明にお
いて酵素的処理とは、酵素を用いてペプチドのクリーベ
イジを行う処理をさす。該処理として具体的には、例え
ばキモトリプシン分解や、Ｖ8 プロテアーゼによるＧｌ
ｕ残基での開裂等が挙げられるが、前記の作用を有する
ものであれば特に限定されるものではない。Next, a chemical and / or enzymatic treatment is performed. The known chemical treatment in the present invention refers to a treatment for cleaving a peptide by a chemical method. Specific examples of the treatment include cleavage with Met residue by CNBr and Tr by CNBr-DMSO-HCl.
Cleavage at the p residue may be mentioned, but it is not particularly limited as long as it has the above-mentioned action. In the present invention, the enzymatic treatment means a treatment of cleaving a peptide using an enzyme. Specific examples the process, and for example chymotrypsin degradation, Gl by V 8 protease
Cleavage at the u residue and the like can be mentioned, but it is not particularly limited as long as it has the above-mentioned action.
【０００８】次に、得られた各フラグメントのαアミノ
基およびＬｙｓのεアミノ基と反応して共有結合を形成
しうる官能基を有する固体と各フラグメントとを反応さ
せ固定化し、トリフルオロ酢酸等を含有する洗浄液で洗
浄を行う。本発明においてアミノ基と反応して共有結合
を形成しうる官能基とは、イミド基、アルデヒド基、シ
アノ基、アセチル基、サクシニル基、マレイル基、イソ
チオシアナート基等が挙げられるが、アミノ基との特異
的反応性の点から、ならびに結合後の特異的切断の目的
からイソチオシアナート基が好適である。また、該官能
基を有する固体とは固体担体をさし、例えば、多孔性ガ
ラス、シリカゲル、ポリスチレン等の材質のものが挙げ
られ、具体的にはＤＩＴＣ−ポリスチレン等が挙げられ
る。Next, the solid having a functional group capable of reacting with the α-amino group of each fragment and the ε-amino group of Lys to form a covalent bond is reacted with each fragment for immobilization, and trifluoroacetic acid, etc. Cleaning is performed with a cleaning liquid containing. In the present invention, the functional group capable of reacting with an amino group to form a covalent bond includes an imide group, an aldehyde group, a cyano group, an acetyl group, a succinyl group, a maleyl group, and an isothiocyanate group. An isothiocyanate group is preferable from the viewpoint of specific reactivity with and from the purpose of specific cleavage after binding. In addition, the solid having the functional group refers to a solid support, and examples thereof include materials such as porous glass, silica gel, and polystyrene, and specifically, DITC-polystyrene and the like.
【０００９】Ｎ末端フラグメントは、αアミノ基がブロ
ックされており、また、Ｌｙｓのεアミノ基がアセチル
化されているので、前記反応により固定化されず、遊離
のＮ末端フラグメントとして洗浄後の液中に溶出する。
これを回収する目的で、遊離のＮ末端フラグメントをア
リルアミン型官能基で誘導体化されたポリマーの膜また
はガラス繊維濾紙上で分取する。本発明においてアミノ
基で誘導体化されたポリマーの膜またはガラス繊維濾紙
とは、例えば、具体的にはポリマーの膜としては、アリ
ルアミンメンブランであるＰＶＤＦ (ポリビニリデンダ
イフルオライド)(Sequelon AA TM、Milligen社)等が、
ガラス繊維濾紙の材料である担体ガラス繊維としては、
アミノプロピルグラス、アミノフェニルグラス等が挙げ
られる。また、ポリマーの膜のサイズは８〜１０ｍｍφ
が良く、フラクションを分取するバイアル内にそのまま
装填でき、そのままシーケンサーのリアクションチェン
バーカートリッジに入る大きさであればよい。Since the α-amino group of the N-terminal fragment is blocked and the ε-amino group of Lys is acetylated, the N-terminal fragment is not immobilized by the above reaction and is a liquid after washing as a free N-terminal fragment. Elutes in.
To recover this, the free N-terminal fragment is fractionated on a membrane of polymer derivatized with allylamine-type functional groups or on glass fiber filter paper. In the present invention, the amino group-derivatized polymer membrane or the glass fiber filter paper is, for example, specifically a polymer membrane such as PVDF (polyvinylidene difluoride) (Sequelon AA ™ , Milligen) which is an allylamine membrane. Company)
As the carrier glass fiber which is the material of the glass fiber filter paper,
Examples include aminopropyl glass and aminophenyl glass. The size of the polymer film is 8 to 10 mmφ.
The size may be such that it can be directly loaded into a vial for fraction collection and can be directly inserted into the reaction chamber cartridge of the sequencer.
【００１０】次に、分取後のポリマーの膜またはガラス
繊維濾紙に水溶性カルボジイミド（ＥＤＣ））を加えて
しばらくの間放置した後、該膜（またはガラス繊維濾
紙）をアセトニトリルで洗浄し、乾燥させる。このと
き、Ｎ末端フラグメント中のＣ末端カルボキシル基もし
くは該フラグメント中のＡｓｐ（アスパラギン酸）およ
び／またはＧｌｕ（グルタミン酸）の側鎖カルボキシル
基を介して膜に固定化される。Next, water-soluble carbodiimide (EDC) is added to the polymer film or glass fiber filter paper after fractionation and left for a while, and then the film (or glass fiber filter paper) is washed with acetonitrile and dried. Let At this time, it is immobilized on the membrane via the C-terminal carboxyl group in the N-terminal fragment or the side chain carboxyl group of Asp (aspartic acid) and / or Glu (glutamic acid) in the fragment.
【００１１】尚、本発明の方法により得られた、該ポリ
マーの膜または該ガラス繊維濾紙上に固定化されたペプ
チドＮ末端フラグメントがアセチル基を有する場合は、
アシルアミノ酸遊離酵素（Acylamino acid-releasing e
nzyme)（宝酒造より市販されているので容易に入手可能
である）により、該ポリマーの膜（または該ガラス繊維
濾紙）に固定化されたペプチドＮ末端フラグメントを処
理する。アシルアミノ酸遊離酵素は、分子の大きいタン
パク質には作用しないことが知られているので、上述の
固定化されたペプチドＮ末端フラグメントのように断片
化されたフラグメントに用いることが可能である。前記
酵素処理の後、該ポリマーの膜（または該ガラス繊維濾
紙）の膜上の酵素残渣を洗浄液（含水アセトニトリルま
たは含水メタノール）により洗い流してから、バイアル
内にそのまま装填して、シーケンサーのリアクションチ
ェンバーカートリッジに挿入することができる。When the peptide N-terminal fragment immobilized on the polymer membrane or the glass fiber filter paper obtained by the method of the present invention has an acetyl group,
Acylamino acid-releasing e
nzyme) (commercially available from Takara Shuzo), the peptide N-terminal fragment immobilized on the membrane of the polymer (or the glass fiber filter paper) is treated. Acyl amino acid-releasing enzymes are known not to act on large proteins and can therefore be used on fragmented fragments such as the immobilized peptide N-terminal fragment described above. After the enzyme treatment, the enzyme residue on the membrane of the polymer membrane (or the glass fiber filter paper) is washed off with a washing solution (hydrous acetonitrile or hydrous methanol), and then loaded into a vial as it is, and a reaction chamber cartridge of a sequencer. Can be inserted into.
【００１２】また、シーケンサーによる分析を行う際の
前処理として、該ポリマーの膜（または該ガラス繊維濾
紙）に固定化されたペプチドＮ末端フラグメントがホル
ミル基および／またはピログルタミル基を有している場
合、それぞれ塩酸あるいはヒドラジン等で処理すること
により、Ｎ末端のαアミノ基のブロックを除去すること
ができる。ここで、ペプチドＮ末端フラグメントがピロ
グルタミル基を有している場合は、エンザイムによる処
理により、Ｎ末端のαアミノ基のブロックを除去するこ
とも可能である。Further, as a pretreatment for analysis by a sequencer, the peptide N-terminal fragment immobilized on the polymer membrane (or the glass fiber filter paper) has a formyl group and / or a pyroglutamyl group. In this case, the block of the α-amino group at the N-terminal can be removed by treating each with hydrochloric acid or hydrazine. Here, when the peptide N-terminal fragment has a pyroglutamyl group, the block of the N-terminal α-amino group can be removed by treatment with an enzyme.
【００１３】[0013]
【実施例】以下、実施例により本発明をさらに詳しく説
明するが、本発明はこの実施例によりなんら限定される
ものではない。 実施例１ 本発明のペプチドＮ末端フラグメント分取方法につい
て、図１および図２を用いて以下に説明する。図１は、
本発明におけるＮ末端フラグメントの分取の各工程の流
れを示す概略図である。図２は、本発明におけるＮ末端
フラグメントの分取および固定化を行なう装置の概略構
成図である。以下、図２の概略構成図に従って説明す
る。まず、Ｎ末端がブロックされた目的のペプチド（タ
ンパク質）の、Ｌｙｓのεアミノ基をアセチルイミダゾ
ールまたは無水酢酸等で室温にて２０分間程度のアセチ
ル化を行った。（無水酢酸を用いた場合、後述のＮ2 パ
ージによりほぼ完全に除去が可能である。）次に、化学
的および／または酵素的処理を常法（Methodin Enzymol
ogy等の文献に記載の方法または各酵素メーカーにより
公知のプロトコールに記載の方法）に従って行った。The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 A method for fractionating a peptide N-terminal fragment of the present invention will be described below with reference to FIGS. 1 and 2. Figure 1
It is a schematic diagram showing the flow of each process of fractionation of the N terminal fragment in the present invention. FIG. 2 is a schematic configuration diagram of an apparatus for sorting and immobilizing the N-terminal fragment in the present invention. Hereinafter, description will be given according to the schematic configuration diagram of FIG. First, the N-terminal blocked peptide (protein) was subjected to acetylation of the ε-amino group of Lys with acetylimidazole or acetic anhydride at room temperature for about 20 minutes. (When acetic anhydride is used, it can be almost completely removed by N 2 purging described later.) Next, chemical and / or enzymatic treatment is performed by a conventional method (Method in Enzymol).
It was performed according to the method described in the literature such as ogy or the method described in the protocol known by each enzyme manufacturer).
【００１４】その後、反応混合物をＤＩＴＣ−ＣＰＧ
（ＤＩＴＣ−ビーズ、シグマ社製、特開平１−２３５６
００号公報参照）に注入し、反応系を窒素置換して、室
温で１時間程度のカップリング反応を行った。これらの
反応は反応最適条件下で行うためにヒーターを有する反
応槽１のカラム内でおこなうことが望ましい。反応終了
後、洗浄液（０．１％トリフルオロ酢酸を含有する５０
％アセトニトリル・２−プロパノール溶液（体積比３／
７）で洗浄した。この時、洗浄液中にはＮ末端のαアミ
ノ基がブロックされたＮ末端フラグメントが溶出するの
で、該洗浄液を回収容器２に回収した。この結果、Ｎ末
端フラグメント以外のフラグメントは固定化された。Thereafter, the reaction mixture was treated with DITC-CPG.
(DITC-Beads, manufactured by Sigma, JP-A-1-2356
No. 00), the reaction system was replaced with nitrogen, and the coupling reaction was carried out at room temperature for about 1 hour. These reactions are preferably carried out in the column of the reaction tank 1 having a heater in order to carry out the reaction under optimum conditions. After completion of the reaction, a washing solution (containing 50% of 0.1% trifluoroacetic acid)
% Acetonitrile / 2-propanol solution (volume ratio 3 /
It was washed in 7). At this time, the N-terminal fragment in which the N-terminal α-amino group was blocked was eluted in the washing liquid, so the washing liquid was collected in the collecting container 2. As a result, fragments other than the N-terminal fragment were immobilized.
【００１５】一方、回収容器２内のＮ末端フラグメント
を含有する溶出液には、化学的および／または酵素的処
理時の試薬バッファー、デタージェント類が混入してい
るので、同回収容器２に対して設けたＮ2 パージライン
４よりＮ2 パージを回収容器２内に送り、ペプチドＮ末
端フラグメントが溶解している溶出液を蒸発・乾燥させ
た。次に、回収容器２をヒートブロック６に入れ５５℃
で保温したのち、さらにＮ2 パージを行って溶媒を除去
した後、室温に戻した。同回収容器２に膜５をいれたま
ま、この中に３０〜５０％アセトニトリル（３０μｌ以
下）を加えた。次に、反応液（0.1M 4−モルホリンエタ
ンスルホン酸（ＭＥＳ）ｐＨ5.0 、15％アセトニトリ
ル、10mg/ml 水溶性カルボジイミド（ＥＤＣ））１５μ
ｌを加えて２０分間放置した。このとき、αアミノ基が
ブロックされたＮ末端フラグメント中のＣ末端カルボキ
シル基もしくは該フラグメント中のＡｓｐ（アスパラギ
ン酸）および／またはＧｌｕ（グルタミン酸）の側鎖カ
ルボキシル基を介してＮ末端フラグメントが膜５に固定
化された。On the other hand, since the eluate containing the N-terminal fragment in the recovery container 2 is contaminated with reagent buffers and detergents at the time of chemical and / or enzymatic treatment, the N 2 purge than N 2 purge line 4 provided Te sent to the collection container 2, evaporated and dried eluate peptide N-terminal fragment is dissolved. Next, the recovery container 2 is put in the heat block 6 and the temperature is 55 ° C.
After the temperature was kept at 1, the mixture was further purged with N 2 to remove the solvent and then returned to room temperature. With the membrane 5 still in the recovery container 2, 30 to 50% acetonitrile (30 μl or less) was added thereto. Next, the reaction solution (0.1 M 4-morpholineethanesulfonic acid (MES) pH 5.0, 15% acetonitrile, 10 mg / ml water-soluble carbodiimide (EDC)) 15 μ
1 was added and left for 20 minutes. At this time, the N-terminal fragment is bound to the membrane 5 through the C-terminal carboxyl group in the N-terminal fragment in which the α-amino group is blocked or the side chain carboxyl group of Asp (aspartic acid) and / or Glu (glutamic acid) in the fragment. Fixed to.
【００１６】次に、膜５をとり出し、メタノールと蒸留
水で交互に数回膜５を洗浄し、リンスまたは乾燥させた
後、前述のアシルアミノ酸遊離酵素（宝酒造製）により
Ｎ末端のアミノ酸を除去した。さらに膜５を洗浄してシ
ーケンサーにアプライした。その結果、アミノ酸配列の
うちＮ末端より２番目からの配列が検出できた。Next, the membrane 5 is taken out, washed with the methanol and distilled water alternately several times, rinsed or dried, and then the N-terminal amino acid is removed by the aforementioned acylamino acid-releasing enzyme (Takara Shuzo). Removed. Further, the membrane 5 was washed and applied to a sequencer. As a result, the second amino acid sequence from the N-terminal could be detected.
【００１７】[0017]
【発明の効果】本発明の分取方法によれば、非常に微量
のサンプルからでもＮ末端フラグメントの分取を行うこ
とが可能で、これを膜に固定化せずにそのまま質量分析
により配列を決定することが可能で、さらに引き続きア
シルアミノ酸遊離酵素で処理を行なってからアミノ酸配
列分析を行うことも可能である。また、フラグメントの
分取と固定化とを同一容器内で迅速に行うとともに、得
られた固定化物をそのままアミノ酸配列分析に用いる分
析機の反応器に取りつけることができるので、コンタミ
ネーションの危険性が非常に少ない上、更にメカニカル
ロスも少ないという利点がある。すなわち、本発明の方
法が提供されることにより、サンプルのＮ末端フラグメ
ントを半自動的に分取し、かつ固定化することが可能と
なった。According to the fractionation method of the present invention, it is possible to fractionate an N-terminal fragment even from a very small amount of sample, and the sequence can be directly analyzed by mass spectrometry without immobilizing it on a membrane. It is possible to determine, and it is also possible to perform subsequent treatment with an acylamino acid-releasing enzyme before amino acid sequence analysis. In addition, since fractionation and immobilization of fragments can be performed rapidly in the same container and the obtained immobilization product can be directly attached to the reactor of the analyzer used for amino acid sequence analysis, there is no risk of contamination. It has the advantage that it is extremely small and also has little mechanical loss. That is, by providing the method of the present invention, it became possible to fractionate and immobilize the N-terminal fragment of a sample semi-automatically.
【図１】図１は、本発明におけるＮ末端フラグメントの
分取の各工程の流れを示す概略図である。FIG. 1 is a schematic diagram showing a flow of each step of fractionating an N-terminal fragment in the present invention.
【図２】図２は、本発明におけるＮ末端フラグメントの
分取および固定化を行なう装置の概略構成図である。FIG. 2 is a schematic configuration diagram of an apparatus for sorting and immobilizing an N-terminal fragment according to the present invention.
１ 反応槽 ２ 回収容器 ３ 反応液送液ライン ４ Ｎ2 パージライン ５ 膜 ６ ヒートブロック1 Reaction Tank 2 Recovery Container 3 Reaction Liquid Delivery Line 4 N 2 Purge Line 5 Membrane 6 Heat Block
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｇ０１Ｎ 33/68 7055−2Ｊ ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Internal reference number FI technical display location G01N 33/68 7055-2J
Claims (1)
ロックされたペプチドにおいて、Ｌｙｓのεアミノ基を
アセチル化し、該アセチル化体を化学的処理および／ま
たは酵素的処理を行い、得られた各フラグメントのαア
ミノ基およびＬｙｓのεアミノ基と反応して共有結合を
形成しうる官能基を有する固体と各フラグメントとを反
応させ、次いで遊離のＮ末端フラグメントをアリルアミ
ン型官能基で誘導体化されたポリマーの膜またはガラス
繊維濾紙上で分取し、分取されたＮ末端フラグメントを
水溶性カルボジイミド等を用いて該Ｎ末端フラグメント
中のカルボキシル基を介して、該ポリマーの膜または該
ガラス繊維濾紙上に共有結合により固定化することを特
徴とする、ペプチドＮ末端フラグメントの分取方法。1. A peptide obtained by acetylating the ε-amino group of Lys in a peptide in which the α-amino group at the amino terminus (N-terminus) is blocked, and chemically and / or enzymatically treating the acetylated product. Each fragment was reacted with a solid having a functional group capable of reacting with the α-amino group of each fragment and the ε-amino group of Lys to form a covalent bond, and then the free N-terminal fragment was derivatized with an allylamine type functional group. The polymer film or the glass fiber is separated on a polymer film or a glass fiber filter paper, and the separated N-terminal fragment is subjected to a carboxyl group in the N-terminal fragment using a water-soluble carbodiimide or the like. A method for fractionating a peptide N-terminal fragment, which comprises immobilizing it on a filter paper by a covalent bond.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP35949592A JP3353278B2 (en) | 1992-12-24 | 1992-12-24 | Method for fractionating peptide N-terminal fragment |
| DE19934344425 DE4344425A1 (en) | 1992-12-24 | 1993-12-24 | Collecting N-terminal peptide fragments of proteins |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP35949592A JP3353278B2 (en) | 1992-12-24 | 1992-12-24 | Method for fractionating peptide N-terminal fragment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH06189784A true JPH06189784A (en) | 1994-07-12 |
| JP3353278B2 JP3353278B2 (en) | 2002-12-03 |
Family
ID=18464800
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP35949592A Expired - Lifetime JP3353278B2 (en) | 1992-12-24 | 1992-12-24 | Method for fractionating peptide N-terminal fragment |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP3353278B2 (en) |
| DE (1) | DE4344425A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005139174A (en) * | 2003-10-16 | 2005-06-02 | Shimadzu Corp | Method for converting protein or peptide to its sulfonic acid derivative |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9609262D0 (en) * | 1996-05-02 | 1996-07-03 | Isis Innovation | Peptide library and method |
| DE60215498T2 (en) | 2001-06-07 | 2007-04-26 | Electrophoretics Ltd., Cobham | METHOD FOR DETERMINING POLYPEPTIDES |
| EP1267170A1 (en) * | 2001-06-07 | 2002-12-18 | Xzillion GmbH & CO.KG | Method for characterising polypeptides |
| US20040253636A1 (en) * | 2001-09-27 | 2004-12-16 | Mikhail Soloviev | Method of protein analysis |
| ATE381018T1 (en) * | 2002-05-10 | 2007-12-15 | Proteome Factory Ag | SOLID PHASE ASSISTED SPECTROSCOPIC AND SPECTROMETRIC ANALYSIS OF COMPLEX BIOPOLYMER MIXTURES |
| EP1361438A1 (en) * | 2002-05-10 | 2003-11-12 | Proteome Factory AG | Solid-phase assisted spectroscopic and spectrometric analysis of complex peptide mixes |
-
1992
- 1992-12-24 JP JP35949592A patent/JP3353278B2/en not_active Expired - Lifetime
-
1993
- 1993-12-24 DE DE19934344425 patent/DE4344425A1/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005139174A (en) * | 2003-10-16 | 2005-06-02 | Shimadzu Corp | Method for converting protein or peptide to its sulfonic acid derivative |
Also Published As
| Publication number | Publication date |
|---|---|
| DE4344425A1 (en) | 1994-06-30 |
| JP3353278B2 (en) | 2002-12-03 |
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