JP6937039B2 - アルキン含有分子の濃縮精製方法 - Google Patents
アルキン含有分子の濃縮精製方法 Download PDFInfo
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- JP6937039B2 JP6937039B2 JP2018542019A JP2018542019A JP6937039B2 JP 6937039 B2 JP6937039 B2 JP 6937039B2 JP 2018542019 A JP2018542019 A JP 2018542019A JP 2018542019 A JP2018542019 A JP 2018542019A JP 6937039 B2 JP6937039 B2 JP 6937039B2
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- Prior art keywords
- alkyne
- silica gel
- silver
- terminal alkyne
- peptide
- Prior art date
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- CRQIYTGXNJRYCL-UHFFFAOYSA-N chloroform;methanol;propan-2-ol Chemical compound OC.CC(C)O.ClC(Cl)Cl CRQIYTGXNJRYCL-UHFFFAOYSA-N 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
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- MQIKJSYMMJWAMP-UHFFFAOYSA-N dicobalt octacarbonyl Chemical group [Co+2].[Co+2].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-].[O+]#[C-] MQIKJSYMMJWAMP-UHFFFAOYSA-N 0.000 description 1
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- 238000005040 ion trap Methods 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- 125000003473 lipid group Chemical group 0.000 description 1
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- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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- 229920002223 polystyrene Polymers 0.000 description 1
- GUUBJKMBDULZTE-UHFFFAOYSA-M potassium;2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid;hydroxide Chemical compound [OH-].[K+].OCCN1CCN(CCS(O)(=O)=O)CC1 GUUBJKMBDULZTE-UHFFFAOYSA-M 0.000 description 1
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- XTUSEBKMEQERQV-UHFFFAOYSA-N propan-2-ol;hydrate Chemical compound O.CC(C)O XTUSEBKMEQERQV-UHFFFAOYSA-N 0.000 description 1
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- 150000003839 salts Chemical class 0.000 description 1
- 125000005372 silanol group Chemical group 0.000 description 1
- SDLBJIZEEMKQKY-UHFFFAOYSA-M silver chlorate Chemical compound [Ag+].[O-]Cl(=O)=O SDLBJIZEEMKQKY-UHFFFAOYSA-M 0.000 description 1
- KKKDGYXNGYJJRX-UHFFFAOYSA-M silver nitrite Chemical compound [Ag+].[O-]N=O KKKDGYXNGYJJRX-UHFFFAOYSA-M 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
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- 229910052979 sodium sulfide Inorganic materials 0.000 description 1
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 description 1
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- 230000001629 suppression Effects 0.000 description 1
- FKHIFSZMMVMEQY-UHFFFAOYSA-N talc Chemical compound [Mg+2].[O-][Si]([O-])=O FKHIFSZMMVMEQY-UHFFFAOYSA-N 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
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- 125000000101 thioether group Chemical group 0.000 description 1
- 150000003568 thioethers Chemical group 0.000 description 1
- PUBSFLYPENQNMY-UHFFFAOYSA-N trimethyl(2-prop-2-ynoxyethyl)azanium Chemical class C[N+](C)(C)CCOCC#C PUBSFLYPENQNMY-UHFFFAOYSA-N 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
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- 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
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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
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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
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/34—Regenerating or reactivating
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- Chemical Kinetics & Catalysis (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Immunology (AREA)
- Pathology (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Description
(1)銀イオンがpKa2〜9の官能基を介して担持された不溶性担体を含有する、液体中の末端アルキン含有分子の濃縮精製用吸着剤。
(2)pKa2〜9の官能基がカルボキシル基、リン酸基又はスルホンアミド基である前記(1)に記載の吸着剤。
(3)前記不溶性担体が、銀イオンがカルボキシル基を介して担持されたシリカゲル系又は有機ポリマー系担体である前記(1)に記載の吸着剤。
(4)前記(1)〜(3)のいずれかに記載の吸着剤を用いた末端アルキン含有分子の濃縮精製用の器具又は装置。
(5)銀イオンがカルボキシル基を介して担持されたシリカゲルと、チオール又はチオウレアで修飾されたシリカゲルが二層構造で用いられている前記(4)に記載の器具又は装置。
(6)複数の層を有し、少なくとも一層が前記(1)〜(3)のいずれかに記載の吸着剤を含有し、及び少なくとも一層が逆相担体を含有する前記(4)又は(5)に記載の器具又は装置。
(7)器具又は装置が固相抽出カラム、キット、フィルター又はチップである前記(4)〜(6)のいずれかに記載の器具又は装置。
(8)前記(1)〜(3)のいずれかに記載の吸着剤に、末端アルキン含有分子を含む液体を接触させて、末端アルキン含有分子を捕捉する工程、及びハロゲン化物イオン、酸又は硫黄含有試薬により溶出する工程を含む、末端アルキン含有分子を濃縮精製する方法。
(9)前記(4)〜(7)のいずれかに記載の器具又は装置を用いる前記(8)に記載の方法。
(10)末端アルキン含有分子を含む液体における溶媒が有機溶媒又は水系溶媒である前記(8)又は(9)に記載の方法。
ガラス製パスツールピペットを用いたアルキン濃縮精製カラムの作成例を示す(図1参照)。
アルキン濃縮精製に適した担体をスクリーニングするため、各種シリカゲル等担体を用いて実施例1と同様にしてカラムを作成し、アルキン含有モデル化合物(N−プロパルギルダンシルアミド)を50%イソプロパノール水溶液に溶解したサンプルを用いて、銀の担持力及びアルキンの濃縮精製効率を調べた。各画分中のモデル化合物の量は、薄層クロマトグラフィー上での蛍光検出により定量し、銀イオンの各画分への溶出は薄層クロマトグラフィー上で4−ジメチルアミノベンジリデンロダニンで染色することで調べた。検討結果を表1に示した。
Normal silica gels:無修飾のシリカゲル;関東化学社製シリカゲル60N(粒子径100−210μm)
C8(octyl):オクチル基修飾シリカゲル;和光純薬社製ワコーゲルTM50C18(破砕状、粒子径40−63μm)
SO3H:スルホン酸修飾シリカゲル;富士シリシア化学社製CHROMATOREX SO3H、MB100−75/200(粒子径75−200μm、平均細孔径100Å)Cyano:シアノプロピル基修飾シリカゲル;SiliCycle社製SiliaBondTMCyano(粒子径40−63μm、平均細孔径60Å、比表面積500m2/g、修飾密度1.38mmol/g)
Diol:富士シリシア化学社製CHROMATOREX Diol、MB100−75/200(粒子径75−200μm、細孔径100Å)
NH2:アミノプロピル修飾シリカゲル;関東化学社製、シリカゲル60(球状)NH2(粒子径40−50μm、平均細孔径64Å、比表面積730m2/g)
−NHCH2CH2NHCH2CH2NH2:関東化学社製R−Cat−Sil TA(修飾密度1.0mmol/g)
Celite 545:バイオタージ社製CeliteTM545
Florisil:US Silica社製フロリジル
COOH:カルボン酸修飾シリカゲル;富士シリシア化学社製CHROMATOREX COOH、MB100−75/200(粒子径75−200μm、平均細孔径100Å)Thiourea:SiliCycle社製SiliaMetSTMThiourea(粒子径40−63μm、平均細孔径60Å、修飾密度1.0mmol/g)
Thiol:東京化成社製3-Mercaptopropyl silica gel(0.5−0.8mmol/g)
Imidazole:SiliCycle社製SiliaMetSTMImidazole(粒子径40−63μm、平均細孔径60Å、修飾密度1.2mmol/g
Piperazine:アルドリッチ社製3-(1-Piperazino)propyl functionalized silica gel(40−75μm、0.8mmol/g)
Phosphine:アルドリッチ社製2-Diphenylphosphinoethyl-functionalized silica gel(40−75μm、0.7mmol/g)
TAAcOH(EDTA型):SiliCycle社製SilliaMetSTMTAAcOH(粒子径40−63μm、平均細孔径60Å、修飾密度0.4mmol/g)
強:銀イオン担持時の素通り画分において、4−ジメチルアミノベンジリデンロダニンの明確な発色反応がない。
中:銀イオン担持時の素通り画分において、4−ジメチルアミノベンジリデンロダニンの発色反応がみられる。
弱:銀イオン担持時の素通り画分において、4−ジメチルアミノベンジリデンロダニンの明確な発色が観察される。
無:銀イオン担持時の素通り画分において、4−ジメチルアミノベンジリデンロダニンの明確な発色が観察され、かつその後のバッファーを用いた洗浄などにおいては発色が観察されない(銀イオンが担持されていないことが疑われる場合)。
良:アルキン化ダンシルアミドは、素通り及び洗浄画分には5%以下、溶出画分において90%以上回収されている。
可:アルキン化ダンシルアミドが、素通り及び洗浄画分に5%以上検出されているが、溶出画分に40%以上回収されている(溶出条件への切り替えに応じてアルキン化ダンシルアミドが溶出している)。
不可:前記以外。アルキン化ダンシルアミドが素通り及び洗浄画分に検出されるとともに、溶出条件に切り替えることで溶出が始まるという挙動が見られない。
強:素通り及び洗浄画分に銀イオンによる4−ジメチルアミノベンジリデンロダニンの明確な発色反応が見られない。
中:素通り及び洗浄画分に4−ジメチルアミノベンジリデンロダニンの発色反応が見られる。
弱:素通り及び洗浄画分に4−ジメチルアミノベンジリデンロダニンの発色反応が強く見られ、速やかに銀イオンが溶出されている。
各種官能基を有するアミノ酸誘導体(Fmoc保護体)の混合物から、アルキン含有アミノ酸を濃縮精製した例を示す(図2参照)。サンプルとしては、7.5mM酢酸アンモニウムを含む50%イソプロパノール−水混合溶媒中に25μMで溶解したものを使用し、前記で説明したアルキン濃縮精製用カラムに通し、同溶媒にて洗浄後、10mMヨウ化ナトリウムにて溶出を行った。検出は、逆相液体クロマトグラフィー及びUV検出器を用いて行った。なお、溶出に用いる方法は、その後の分析に影響を与えないものを適宜選択することができる。
前記実施例において、ペプチドに含まれうる各種官能基存在下においてもアルキン含有分子を濃縮精製できることを示した。本実施例では、アルキン含有モデルペプチドを濃縮精製した例を示す。
本実施例では、本発明の方法が複雑な脂質混合物(培養細胞の脂質抽出物)の中から、アルキン化リン脂質(Hexynoyl PE, from avanti polar lipids)を濃縮精製できることを示す。
本実施例においては、アルキン化分子の細胞内における代謝産物を、それら代謝産物に代謝的に組み込まれるアルキンを利用して濃縮精製した例を示す。アルキンはサイズが小さいため、アルキンを導入した分子は、多くの場合、細胞内の代謝酵素等にも内在性の分子と同様に代謝される。よって、アルキンを導入した代謝産物の前駆体を細胞などに投与し、その代謝産物を本手法により濃縮精製し、質量分析などの手法により構造解析を行うことが可能である。ここでは、脂質前駆体となるコリンの代謝産物の濃縮精製を行った例を示す。
生理活性物質の作用機序の解明には、その生理活性物質がどのようなタンパク質群に結合するのか、そのタンパク質上のどの部位に結合して作用しているのかを明らかにすることが重要である。また、タンパク質の機能にはアセチル化や脂質修飾などの翻訳後修飾が重要な役割を担うことが知られており、翻訳後修飾の解析はタンパク質の機能を解明する上で重要である。通常、結合タンパク質群の同定や化合物の結合部位の同定、翻訳後修飾部位の同定には質量分析法が用いられる。一般的な流れとしては、細胞抽出物など試料をトリプシンなどの酵素で消化し、ペプチド断片とし、得られたペプチド断片の質量電荷比及びフラグメンテーションのパターンをデータベースに照合することで、タンパク質及びペプチド配列を同定する。多くの場合、このような解析では細胞抽出物などのような複雑な混合物を用いられるが、複雑な混合物中ではイオン化抑制等が起こるため、量が少ないペプチド・タンパク質の同定は困難である。本発明は、複雑な混合物中からアルキンを持つペプチドを選択的に精製することを可能にし、イオン化抑制などの問題点の解決に資する。本実施例では、代表的な実験例として、細胞抽出物のトリプシン消化物にアルキン化ペプチドを混合し、そのアルキン化ペプチドを精製する例を示す。
通常、細胞抽出物などをトリプシンなどのプロテアーゼで分解して得られるペプチド混合物は、逆相担体を用いて脱塩処理を行った上で質量分析法による解析が行われる。微量サンプルを扱う場合、工程数が増えるとマイクロチューブなどの容器への吸着が問題となりうる。このような問題に対して、アルキン化ペプチドの濃縮精製の工程とその後の逆相担体の脱塩処理を一つのスピンカラムで行うことが、吸着によるサンプルのロスを抑えるために有効である。本実施例では、銀担持担体と逆相担体を二層構造としたスピンカラムを用いたアルキン含有分子の濃縮精製例を示す。また、銀を担持する担体として、シリカゲル型の担体だけでなく、カルボン酸修飾されたポリマー型の不溶性担体も利用可能であることを示す。代表的な実験例として、HeLa細胞抽出物をトリプシン消化したペプチド混合物から、アルキン化ペプチドを濃縮精製し、LC−MS/MS分析を行った実験例を示す。
Claims (13)
- 銀イオンがpKa2〜9の官能基を介して担持された不溶性担体を含有する、液体中の末端アルキン含有分子の濃縮精製用吸着剤であって、前記液体の溶媒が水系溶媒である濃縮精製用吸着剤。
- 前記末端アルキン含有分子が、塩基性化合物、アミノ酸誘導体、脂質代謝物、水溶性代謝物、ペプチド及び複合脂質から選ばれる少なくとも1種である請求項1記載の吸着剤。
- 前記液体が細胞抽出物又は細胞酵素消化物を含む請求項1又は2記載の吸着剤。
- pKa2〜9の官能基がカルボキシル基、リン酸基又はスルホンアミド基である請求項1〜3のいずれか1項に記載の吸着剤。
- 前記不溶性担体が、銀イオンがカルボキシル基を介して担持されたシリカゲル系又は有機ポリマー系担体である請求項1〜4のいずれか1項に記載の吸着剤。
- 請求項1〜5のいずれか1項に記載の吸着剤を用いた末端アルキン含有分子の濃縮精製用の器具又は装置。
- 銀イオンがカルボキシル基を介して担持されたシリカゲルと、チオール又はチオウレアで修飾されたシリカゲルが二層構造で用いられている請求項6記載の器具又は装置。
- 複数の層を有し、少なくとも一層が請求項1〜5のいずれか1項に記載の吸着剤を含有し、及び少なくとも一層が逆相担体を含有する請求項6又は7記載の器具又は装置。
- 器具又は装置が固相抽出カラム、キット、フィルター又はチップである請求項6〜8のいずれか1項に記載の器具又は装置。
- 請求項1〜5のいずれか1項に記載の吸着剤に、末端アルキン含有分子を含む液体を接触させて、末端アルキン含有分子を捕捉する工程、及びハロゲン化物イオン、酸又は硫黄含有試薬により溶出する工程を含む、末端アルキン含有分子を濃縮精製する方法であって、前記液体の溶媒が水系溶媒である方法。
- 前記末端アルキン含有分子が、塩基性化合物、アミノ酸誘導体、脂質代謝物、水溶性代謝物、ペプチド及び複合脂質から選ばれる少なくとも1種である請求項10記載の方法。
- 前記液体が細胞抽出物又は細胞酵素消化物を含む請求項10又は11記載の方法。
- 請求項6〜9のいずれか1項に記載の器具又は装置を用いる請求項10〜12のいずれか1項に記載の方法。
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