JP5681112B2 - セラミックハイドロキシアパタイトでの分画による組成物中のリン酸化ペプチドの濃縮 - Google Patents
セラミックハイドロキシアパタイトでの分画による組成物中のリン酸化ペプチドの濃縮 Download PDFInfo
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- 239000000203 mixture Substances 0.000 title claims description 40
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 3
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- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 2
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- 229910000403 monosodium phosphate Inorganic materials 0.000 description 2
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
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- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
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- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
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- 108010089430 Phosphoproteins Proteins 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
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- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/04—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium
- B01J20/048—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium containing phosphorus, e.g. phosphates, apatites, hydroxyapatites
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3847—Multimodal interactions
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/282—Porous sorbents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/165—Extraction; Separation; Purification by chromatography mixed-mode chromatography
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- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Peptides Or Proteins (AREA)
Description
本出願は、2008年11月14日付で出願された合衆国特許仮出願番号第61/114801号の利益を主張し、その内容を本明細書中に援用する。
1.本発明の分野
リン酸化ペプチドの分析は、タンパク質機能と生物システムの研究において使用される。タンパク質のリン酸化は、タンパク質の翻訳後修飾中に起こり、多くの場合、タンパク質構造及び機能の保存と調整に重要である。酵素的に触媒されたリン酸化及び脱リン酸化は、生きた細胞の重要な制御的機能であり、そして細胞増殖、発生、及び分化、シグナル伝達、神経活性、細胞骨格の組織化、プログラムされた細胞死、並びに遺伝子発現に寄与している。よって、タンパク質の消化と得られたペプチドの分析によって実施されるリン酸化部位の決定は、複雑な生物システム及び疾患の発現を理解するうえで重要な要素である。しかしながら、リン酸化されたタンパク質やペプチドの研究は、体液中のこれらの種が比較的少量であることによって難しくなっている。そのため、リン酸化種の選択的濃縮を実現するために、様々な方法が開発されてきた。
「ペプチド」という用語は、通常、最大30個のアミノ酸を含有しているアミノ酸のオリゴマーを意味するために本明細書中で使用される。特定の態様において本願発明が対象にすることが好ましいペプチドの質量対電荷比(M/Z)は、約500〜約5000の範囲内、特定の他の実施形態において約500〜約3000の範囲内、特定の他の実施形態において約1000〜約3000の範囲内である。「リン酸化ペプチド」及び「リン酸化ペプチド」という用語は、分子の骨格を形成するアミノ酸鎖に化学的に結合した少なくとも1つのホスホリル基を担持するペプチドを意味するために互換的に使用される。組成物で本願発明によって濃縮され得るリン酸化ペプチドは、単一のホスホリル基が結合したペプチド、並びに2以上のホスホリル基が結合したペプチドを含んでいる。下記にさらに説明し、そして例示するとおり、異なるリン酸化度の(すなわち、1分子あたりの結合したホスホリル基の数が異なる)リン酸化ペプチドは、本発明のプロセスによってそれらのリン酸化度に基づいて互いに分離され得る。
セラミックハイドロキシアパタイトI型、粒度20マイクロメートルを、すべての割合(%)が重量による下記のバッファー溶液と共に使用した:
結合バッファー
40mMのTris、pH9
25mMのNaH2PO4
(尿素、チオ尿素、又はCHAPS不含)
低ストリンジェンシー溶出バッファー
40mMのTris、pH9
25mMのNaH2PO4
500mMのNaCl
(尿素、チオ尿素、又はCHAPS不含)
高ストリンジェンシー溶出バッファー
500mMのNaH2PO4、pH7.5
(尿素、チオ尿素、又はCHAPS不含)
*CHAPS:3‐[(3‐コールアミドプロピル)ジメチルアンモニオ]‐1‐プロパンスルホン酸
次に、上清を回収し、さらに100μLの量の高ストリンジェンシー溶出バッファーを加え、得られた混合物をボルテックスし、遠心機にかけ、そして、上清を高ストリンジェンシー溶出液として最初の上清と一緒に貯めておいた。次に、貯めておいた素通り画分と2種類の貯めておいた溶出液を、留去によって濃縮し、そして、3種類すべてを10%のトリフルオロ酢酸を用いてpH3.0に調整した。
実施例2
実施例3
実施例4
実施例5:比較に関する実施例
実施例6
Claims (4)
- リン酸化ペプチドと非リン酸化ペプチドを含んでなる混合物を分画して、前記リン酸化ペプチドが濃縮された生成物溶液を製造する方法であって、以下のステップ:
(a)pH8〜10のアルカリ性結合バッファー溶液中、前記混合物をセラミックハイドロキシアパタイトと接触させて、前記混合物中のリン酸化ペプチドを前記非リン酸化ペプチドと比べて優先的に前記セラミックハイドロキシアパタイトに結合させ、ここで前記セラミックハイドロキシアパタイトを通過した前記混合物の残留物を、素通り画分と規定し、そして
(b)そのようにして結合した前記リン酸化ペプチドを、アルカリ性溶出バッファーを用いて前記セラミックハイドロキシアパタイトから前記生成物溶液として溶出液中に溶出すること、を含み、ここでステップ(b)を、(i)低ストリンジェンシーアルカリ性溶出バッファーを用いて溶出を実施して、最初の溶出液を生じる第一段階、そして(ii)前記第一段階に続いて、高ストリンジェンシーアルカリ性溶出バッファーを用いて溶出を実施して、2番目の溶出液を生じる第二段階、を含んでなる複数の段階により実施し、前記最初の溶出液では、前記素通り画分及び前記2番目の溶出液に比べて一リン酸化ペプチドが濃縮されており、そして前記2番目の溶出液では、前記素通り画分及び前記最初の溶出液に比べて多重リン酸化ペプチドが濃縮され、ここで低ストリンジェンシーアルカリ性溶出バッファーは高ストリンジェンシーアルカリ性溶出バッファーよりも高いイオン強度を有する、
前記方法。 - 前記セラミックハイドロキシアパタイトを、塩化カルシウム溶液とのインキュベーションによって前処理する、請求項1に記載の方法。
- 前記塩化カルシウム溶液が、ほぼ中性のpHにて30mM〜300mMの塩化カルシウムを含有する水性溶液である、請求項2に記載の方法。
- アルカリ性結合バッファーが、40mMのTris、pH9および25mMのNaH2PO4を含んでなり;低ストリンジェンシーアルカリ性溶出バッファーが40mMのTris、pH9、25mMのNaH2PO4および500mMのNaClを含んでなり;且つ高ストリンジェンシーアルカリ性溶出バッファーが500mMのNaH2PO4、pH7.5を含んでなる、請求項1に記載の方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11480108P | 2008-11-14 | 2008-11-14 | |
US61/114,801 | 2008-11-14 | ||
PCT/US2009/064116 WO2010056797A1 (en) | 2008-11-14 | 2009-11-12 | Phosphopeptide enrichment of compositions by fractionation on ceramic hydroxyapatite |
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Publication Number | Publication Date |
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JP2012508754A JP2012508754A (ja) | 2012-04-12 |
JP5681112B2 true JP5681112B2 (ja) | 2015-03-04 |
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EP2349316A4 (en) | 2012-07-04 |
US8907059B2 (en) | 2014-12-09 |
WO2010056797A1 (en) | 2010-05-20 |
CA2743499C (en) | 2017-04-25 |
EP2349316B1 (en) | 2014-10-29 |
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CA2743499A1 (en) | 2010-05-20 |
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