JP2007322293A - De novo sequence analysis method, analysis software, storage medium storing analysis software, and reagent kit - Google Patents
De novo sequence analysis method, analysis software, storage medium storing analysis software, and reagent kit Download PDFInfo
- Publication number
- JP2007322293A JP2007322293A JP2006154148A JP2006154148A JP2007322293A JP 2007322293 A JP2007322293 A JP 2007322293A JP 2006154148 A JP2006154148 A JP 2006154148A JP 2006154148 A JP2006154148 A JP 2006154148A JP 2007322293 A JP2007322293 A JP 2007322293A
- Authority
- JP
- Japan
- Prior art keywords
- sequence analysis
- novo
- novo sequence
- analysis method
- mass
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000012300 Sequence Analysis Methods 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 title claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 8
- 238000001819 mass spectrum Methods 0.000 claims abstract description 12
- 108090000790 Enzymes Proteins 0.000 claims abstract description 9
- 102000004190 Enzymes Human genes 0.000 claims abstract description 9
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical group CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 15
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 229940088598 enzyme Drugs 0.000 claims description 7
- HKOOXMFOFWEVGF-UHFFFAOYSA-N phenylhydrazine Chemical compound NNC1=CC=CC=C1 HKOOXMFOFWEVGF-UHFFFAOYSA-N 0.000 claims description 7
- 229940067157 phenylhydrazine Drugs 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000003776 cleavage reaction Methods 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 230000007017 scission Effects 0.000 claims description 6
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 4
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 claims description 4
- 238000009833 condensation Methods 0.000 claims description 4
- 230000005494 condensation Effects 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 108010053229 Lysyl endopeptidase Proteins 0.000 claims description 3
- 108090000317 Chymotrypsin Proteins 0.000 claims description 2
- 108090001109 Thermolysin Proteins 0.000 claims description 2
- 108090000631 Trypsin Proteins 0.000 claims description 2
- 102000004142 Trypsin Human genes 0.000 claims description 2
- 150000001408 amides Chemical class 0.000 claims description 2
- 229960002376 chymotrypsin Drugs 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 239000012588 trypsin Substances 0.000 claims description 2
- 238000012163 sequencing technique Methods 0.000 claims 1
- 125000000524 functional group Chemical group 0.000 abstract description 7
- 210000004899 c-terminal region Anatomy 0.000 abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 5
- 239000007788 liquid Substances 0.000 abstract description 5
- 101001018085 Lysobacter enzymogenes Lysyl endopeptidase Proteins 0.000 abstract description 4
- 108091005804 Peptidases Proteins 0.000 abstract 1
- 102000035195 Peptidases Human genes 0.000 abstract 1
- 238000010494 dissociation reaction Methods 0.000 abstract 1
- 230000005593 dissociations Effects 0.000 abstract 1
- 230000006698 induction Effects 0.000 abstract 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 abstract 1
- 235000019833 protease Nutrition 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 10
- 238000006482 condensation reaction Methods 0.000 description 6
- 102000007079 Peptide Fragments Human genes 0.000 description 5
- 108010033276 Peptide Fragments Proteins 0.000 description 5
- 238000003277 amino acid sequence analysis Methods 0.000 description 5
- 230000006862 enzymatic digestion Effects 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
Description
本発明は生化学分野におけるプロテオミックスの中でデータベースが存在しないたんぱく質,ペプチドについてアミノ酸配列解析を行うデノボシーケンスにおいて正確にアミノ酸配列解析できるツールに関するものである。 The present invention relates to a tool capable of accurate amino acid sequence analysis in a de novo sequence for performing amino acid sequence analysis on proteins and peptides that do not have a database among proteomics in the biochemical field.
タンパク質の同定やアミノ酸配列解析は、目的のタンパク質を酵素消化して数から数十アミノ酸残基のペプチドフラグメントにしてから、衝突誘起解裂を備えた液体クロマトグラフ質量分析装置により、得られたペプチドフラグメントのマススペクトルの質量数情報と遺伝子情報をもとに作成させたデータベースから合致している数学的値を基に行われてきた。遺伝子的によく研究されているヒトやマウスなどの生物種は、データベースが充実しているが、他の大部分の生物種はデータ登録数が少なくデータベースとして機能していない。もしくはまったくデータベースとして存在していない。データベースが存在しないと衝突誘起解裂を備えた液体クロマトグラフ質量分析装置において得られたマススペクトルだけでは、ペプチドを衝突誘起解裂した後、N末端を有するb系列とC末端を有するy系列の判別が困難になり正確にアミノ酸配列解析は行えない。 For protein identification and amino acid sequence analysis, the target protein is enzymatically digested into peptide fragments of several to several tens of amino acid residues, and then the peptide obtained by a liquid chromatograph mass spectrometer equipped with collision-induced cleavage is used. This has been done on the basis of mathematical values that match from the database created based on the mass number information and gene information of the mass spectrum of the fragment. Biological species such as humans and mice, which are well studied genetically, have a well-developed database, but most other species have few data registrations and do not function as databases. Or it does not exist as a database at all. In the absence of a database, the mass spectrum obtained in a liquid chromatograph mass spectrometer equipped with collision-induced cleavage alone can be used for the b-series having an N-terminus and the y-series having a C-terminus after collision-induced cleavage of the peptide. Discrimination becomes difficult and the amino acid sequence cannot be analyzed accurately.
一般的にデノボシーケンス解析は、衝突誘起解裂を備えた液体クロマトグラフ質量分析計より得られたマススペクトルの質量数情報を基にN末端を含むb系列とC末端を含むy系列の値を区別することなくアミノ酸配列を当てはめていく。このためアミノ酸配列の可能性が多くなり、正確なアミノ酸配列を決定できない。本発明では、タンパク質を酵素消化するさいに縮合反応を優先する酵素選択と反応条件を設定することにより、C末端側に選択的に別の官能基を導入することにより、普通の酵素消化した脱水反応と比較して質量数的に明らかに違いが分かるようになる。この反応を用いることでy系列とb系列を明確に識別することを可能にし、プログラムされたソフトウェアを使って簡単にデノボシーケンスを行うための手法である。 In general, de novo sequence analysis is based on the mass number information of the mass spectrum obtained from a liquid chromatograph mass spectrometer equipped with collision-induced cleavage, and the values of the b series including the N terminus and the y series including the C terminus. The amino acid sequence is applied without distinction. For this reason, the possibility of an amino acid sequence increases, and an accurate amino acid sequence cannot be determined. In the present invention, when enzymatic digestion of a protein is performed, an enzyme selection and a reaction condition that give priority to the condensation reaction are set, and by introducing another functional group selectively on the C-terminal side, dehydration obtained by ordinary enzyme digestion is performed. Compared with the reaction, the difference in mass number becomes clear. By using this reaction, it is possible to clearly distinguish the y series and the b series, and this is a technique for easily performing a de novo sequence using programmed software.
測定すべきタンパク質を酵素消化するさいに、すでに知られている縮合効率の高いリジルエンドペプチダーゼ(Lys−C)酵素を選択し、反応条件は、有機溶媒(ジメチルホルムアミド)高存在下で、酵素反応の至適pHを縮合反応に有利となるようにすこし酸性側にシフトし、基質の10倍以上の導入物質を添加することで、酵素消化するとき縮合反応をさせる。これによりC末端側に質量数の大きな官能基を導入する。導入後、質量分析計で測定するとy系列(官能基の導入されていない)と官能基が導入されているb系列を質量分析計の質量数から簡単に判別できる。 For enzymatic digestion of the protein to be measured, a known lysyl endopeptidase (Lys-C) enzyme with high condensation efficiency is selected, and the reaction conditions are an enzyme reaction in the presence of a high organic solvent (dimethylformamide). The optimum pH is slightly shifted to the acidic side so as to be advantageous for the condensation reaction, and the introduction reaction is carried out by 10 times or more of the substrate so that the condensation reaction is carried out during the enzymatic digestion. This introduces a functional group having a large mass number on the C-terminal side. After introduction, when measured with a mass spectrometer, the y series (with no functional group introduced) and the b series with a functional group introduced can be easily distinguished from the mass number of the mass spectrometer.
本発明により、データベースに依存しないデノボシーケンス解析が正確にかつ短時間に行えるようになる。 According to the present invention, de novo sequence analysis independent of a database can be performed accurately and in a short time.
本発明の一実施例を図面を用いて説明する。 An embodiment of the present invention will be described with reference to the drawings.
図1はデノボシーケンス解析反応手順と縮合反応試薬キットの構成図である。タンパク質の入ったチューブ5に試薬キットの有機溶媒(ジメチルホルムアミド)1を25〜75%の範囲で加える、次に試薬キットトリス緩衝液2を75〜25%の範囲で加える。次に試薬キット導入物質(フェニルヒドラジン)3をタンパク質量の約10〜100倍加える。よく攪拌した後試薬キット酵素(Lys−C)4を2〜20μg加え37℃に保ち酵素反応を4時間から16時間行う。この反応キットおよび反応条件により図2のように
H3O+(OH)の代わりに導入基フェニルヒドラジンがペプチド結合(N−C)切断後C末端側に導入される。酵素消化されたペプチドフラグメントは、衝突誘起解裂を備えた液体クロマトグラフ質量分析計でアミノ酸配列解析を行う。図3は、アミノ酸8残基で構成されたペプチドフラグメントのマススペクトルの例を示す。質量分析の衝突誘起気解裂でペプチドフラグメントを解析すると無作為にペプチド結合が切れてマススペクトルのパターンからはN末端を有するb系列とC末端を有するy系列を明確に判別することは困難であるが、導入基フェニルヒドラジンを導入した反応では、C末端側でフェニルヒドラジンの質量数106だけシフトしたマススペクトルが確認される。図4は、質量分析計によって同じ測定試料から酵素消化法を変えて得られた質量数リストである。図5は、図4の2つの質量数リストからデノボシーケンス解析する方法のフローチャートである。図4の酵素消化の質量数リストの最初の値からデノボ酵素消化の質量数リストの値と比較して元の値より質量数で導入基の106シフトしている値を探し出す。それを最後の値まで行い抽出された値がy系列の候補となる。同様に2つのリストから同一の質量数を抽出することでb系列の候補を探しだす。また上記の条件に合わない値は、溶媒やインターナルフラグメントに由来しているイオンとして分類できる。図6はb系列の候補として抽出されたリストとb系列の候補として抽出されたリストである。大きな値から近接した小さな値を引くことでその差がアミノ酸に対応している。この結果からSFL(I)DSGYRのアミノ酸配列解析が可能となる。
FIG. 1 is a configuration diagram of a de novo sequence analysis reaction procedure and a condensation reaction reagent kit. The reagent kit organic solvent (dimethylformamide) 1 is added in the range of 25-75% to the
上記において、縮合効果を有する酵素としては、リジルエンドペプチターゼ,トリプシン,キモトリプシン,サーモライシンの中から選ばれた少なくとも1種であっても同様の効果を得ることができる。 In the above, the same effect can be obtained even if the enzyme having a condensation effect is at least one selected from lysyl endopeptidase, trypsin, chymotrypsin, and thermolysin.
また、導入物質は、アミド,アニリン,ヒドラジン,フェニルヒドラジンの中から選ばれた少なくとも1種を有する物質であっても良い。 Further, the introduced substance may be a substance having at least one selected from amide, aniline, hydrazine, and phenylhydrazine.
また、有機溶媒はジメチルホルムアミド,酢酸エチル,アセトニトリルであっても良い。 The organic solvent may be dimethylformamide, ethyl acetate, or acetonitrile.
1…有機溶媒(ジメチルホルムアミド)、2…pH緩衝液(トリスバファー)、3…導入基用溶液(フェニルヒドラジン)、4…酵素(Lys−C)、5…反応用チューブ。
DESCRIPTION OF
Claims (7)
該デノボ酵素消化を行った試料を衝突誘起解裂機能を備えた質量分析装置を用いてマススペクトルを取得するステップと、
該ステップで得られたマススペクトルと、通常の酵素消化を行った試料を衝突誘起解裂機能を備えた質量分析装置を用いて得られたマススペクトルと比較し、前記導入物質の質量数だけシフトしているマススペクトルを抽出し、抽出された値をy系列の候補とするステップと、
を含むことを特徴とするデノボシーケンス解析方法。 A de novo enzyme digestion step in which an enzyme having a condensation effect is used to enzymatically digest a protein to be measured, and in the presence of an organic solvent, an introduction substance more than 10 times the substrate is added to perform the enzyme digestion;
Obtaining a mass spectrum of the sample subjected to de novo enzyme digestion using a mass spectrometer having a collision-induced cleavage function;
The mass spectrum obtained in this step is compared with the mass spectrum obtained by using a mass spectrometer equipped with a collision-induced cleaving function for a sample subjected to normal enzyme digestion, and shifted by the mass number of the introduced substance. Extracting a mass spectrum that is being performed, and setting the extracted value as a candidate for a y-series;
A de novo sequence analysis method comprising:
前記縮合効果を有する酵素が、リジルエンドペプチターゼ,トリプシン,キモトリプシン,サーモライシンの中から選ばれた少なくとも1種であることを特徴とするデノボシーケンス解析方法。 In claim 1,
The de novo sequence analysis method, wherein the enzyme having the condensation effect is at least one selected from lysyl endopeptidase, trypsin, chymotrypsin, and thermolysin.
前記導入物質が、アミド,アニリン,ヒドラジン,フェニルヒドラジンの中から選ばれた少なくとも1種を有する物質であることを特徴とするデノボシーケンス解析方法。 In claim 1 or 2,
The de novo sequence analysis method, wherein the introduced substance is a substance having at least one selected from amide, aniline, hydrazine, and phenylhydrazine.
前記有機溶媒がジメチルホルムアミド,酢酸エチル,アセトニトリルのいずれかであることを特徴とするデノボシーケンス解析方法。 In any one of Claims 1-3,
The de novo sequence analysis method, wherein the organic solvent is dimethylformamide, ethyl acetate, or acetonitrile.
A storage medium storing the de novo sequence analysis software according to claim 6.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2006154148A JP4832168B2 (en) | 2006-06-02 | 2006-06-02 | De novo sequence analysis method, analysis software, storage medium storing analysis software, reagent kit |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2006154148A JP4832168B2 (en) | 2006-06-02 | 2006-06-02 | De novo sequence analysis method, analysis software, storage medium storing analysis software, reagent kit |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2007322293A true JP2007322293A (en) | 2007-12-13 |
JP4832168B2 JP4832168B2 (en) | 2011-12-07 |
Family
ID=38855246
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2006154148A Expired - Fee Related JP4832168B2 (en) | 2006-06-02 | 2006-06-02 | De novo sequence analysis method, analysis software, storage medium storing analysis software, reagent kit |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP4832168B2 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2014052331A (en) * | 2012-09-10 | 2014-03-20 | Shimadzu Corp | Analysis method and apparatus for amino acid sequence |
JP2021534408A (en) * | 2018-08-17 | 2021-12-09 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | Methods for DE NOVO Protein Sequencing |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004219418A (en) * | 2003-01-13 | 2004-08-05 | Agilent Technol Inc | Method of selecting n-terminal peptide and c-terminal peptide in proteomics |
JP2004294431A (en) * | 2003-03-11 | 2004-10-21 | Shimadzu Corp | Method of determining amino acid sequence of peptide |
JP2005139174A (en) * | 2003-10-16 | 2005-06-02 | Shimadzu Corp | Method for converting protein or peptide to its sulfonic acid derivative |
-
2006
- 2006-06-02 JP JP2006154148A patent/JP4832168B2/en not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004219418A (en) * | 2003-01-13 | 2004-08-05 | Agilent Technol Inc | Method of selecting n-terminal peptide and c-terminal peptide in proteomics |
JP2004294431A (en) * | 2003-03-11 | 2004-10-21 | Shimadzu Corp | Method of determining amino acid sequence of peptide |
JP2005139174A (en) * | 2003-10-16 | 2005-06-02 | Shimadzu Corp | Method for converting protein or peptide to its sulfonic acid derivative |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2014052331A (en) * | 2012-09-10 | 2014-03-20 | Shimadzu Corp | Analysis method and apparatus for amino acid sequence |
JP2021534408A (en) * | 2018-08-17 | 2021-12-09 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | Methods for DE NOVO Protein Sequencing |
US12000840B2 (en) | 2018-08-17 | 2024-06-04 | Regeneron Pharmaceuticals, Inc. | Methods for de novo protein sequencing |
Also Published As
Publication number | Publication date |
---|---|
JP4832168B2 (en) | 2011-12-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Parker et al. | Forensic proteomics | |
Na et al. | Discovery of noncanonical translation initiation sites through mass spectrometric analysis of protein N termini | |
US6379970B1 (en) | Analysis of differential protein expression | |
Lothrop et al. | Deciphering post-translational modification codes | |
Lubec et al. | Searching for hypothetical proteins: theory and practice based upon original data and literature | |
Knight et al. | Phosphospecific proteolysis for mapping sites of protein phosphorylation | |
Beaudette et al. | Proteomic techniques to probe the ubiquitin landscape | |
WO2010065322A1 (en) | Concurrent identification of multitudes of polypeptides | |
US20050048564A1 (en) | Protein expression profile database | |
Li et al. | Improved sequence variant analysis strategy by automated false positive removal | |
KR100805775B1 (en) | An additive scoring method for modified polypeptide | |
Brown et al. | Direct seminal fluid identification by protease‐free high‐resolution mass spectrometry | |
Jouber et al. | Identification by mass spectrometry of two‐dimensional gel electrophoresis‐separated proteins extracted from lager brewing yeast | |
Schrader et al. | The process chain for peptidomic biomarker discovery | |
JP4832168B2 (en) | De novo sequence analysis method, analysis software, storage medium storing analysis software, reagent kit | |
WO2008011379A4 (en) | Oxidized apoa1 determination by mass spectrometry | |
US20140156206A1 (en) | Detection and Quantification of Polypeptides Using Mass Spectrometry | |
Boudier-Lemosquet et al. | Introducing protein deamidation: Landmark discoveries, societal outreach, and tentative priming workflow to address deamidation | |
James | Chips for proteomics: a new tool or just hype? | |
Löhr et al. | Proteomics in pancreatic disease | |
US20200363423A1 (en) | Mistranslation method for assessing the effects of amino acid substitutions on protein stability and function | |
JP4614960B2 (en) | Testing the amino acid sequence of peptides by isotopic ratio | |
WO2017162888A1 (en) | Universal mrm tags for protein quantification | |
Wu et al. | Analysis of protein phosphorylation using mass spectrometry | |
Kuppusamy et al. | “Omics” approaches to determine protease degradomes in complex biological matrices |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20090520 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20090520 |
|
A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20110609 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20110621 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20110803 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20110823 |
|
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20110920 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 4832168 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140930 Year of fee payment: 3 |
|
S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
LAPS | Cancellation because of no payment of annual fees |