JPWO2015125216A1 - 質量分析を用いたタンパク質の検出方法 - Google Patents
質量分析を用いたタンパク質の検出方法 Download PDFInfo
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- 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
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- 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
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Abstract
Description
(条件1)同じアミノ酸配列が他のタンパク質中に存在しないこと;
(条件2)配列中にシステインを含まないこと;
(条件3)配列のアミノ酸の数が8〜20であること;
(条件4)配列のN末端がグルタミンまたはアスパラギン以外であること;を満たすものを好適な候補ペプチドとした場合、以下のペプチドを選択することができる。
GLGTDEDAIISVLAYR(配列番号3、配列番号1のアミノ酸配列のアミノ酸番号29−44に相当);
ISQTYQQQYGR(配列番号4、配列番号1のアミノ酸配列のアミノ酸番号124−134に相当);
SDTSFMFQR(配列番号5、配列番号1のアミノ酸配列のアミノ酸番号142−150に相当);
VLVSLSAGGR(配列番号6、配列番号1のアミノ酸配列のアミノ酸番号151−160に相当);
DEGNYLDDALVR(配列番号7、配列番号1のアミノ酸配列のアミノ酸番号161−172に相当);
SETSGSFEDALLAIVK(配列番号8、配列番号1のアミノ酸配列のアミノ酸番号226−241に相当);
GLGTDDNTLIR(配列番号9、配列番号1のアミノ酸配列のアミノ酸番号260−270に相当);
AEIDMLDIR(配列番号10、配列番号1のアミノ酸配列のアミノ酸番号276−284に相当);
GDTSGDYR(配列番号11、配列番号1のアミノ酸配列のアミノ酸番号301−308に相当)。
[分析対象ペプチドの選定]
本発明の方法により、卵巣癌のバイオマーカーとして期待される、血清中に微量に含まれるアネキシン A4(ANXA4)(配列番号1)の半定量的な検出を行った。
市販のヒト血清(製品コード14−402E、ロンザジャパン)に対して、大腸菌の発現系により得た精製リコンビナントタンパク質ANXA4を3mg/mLの濃度で10%量添加し、それを上記の血清にて段階希釈することで、ANXA4の濃度が300、150、75、38、19、9、及び5μg/mLの血清試料をそれぞれ調製した。血清試料は56℃、40分の加熱処理により非働化し、さらに0.44μmフィルター(ウルトラフリー、ミリポア社)にて清澄化した。粉末状のトリプシン(製品コード203−09893、和光純薬)を1mmol/Lの塩酸溶液にて10mg/mLの濃度に溶解し、トリプシン溶液とした。
本発明による前処理工程を以下に説明する。上述のように調製した血清試料各100μLを1.5mL容量のエッペンドルフチューブに取り、トリプシン溶液2μLを添加し、37℃で16時間反応させた(血清試料100μLに対してトリプシン約20μg)。反応後の試料を限外濾過フィルター(Amicon 10,000MWCOフィルター、ミリポア社)の上部に移し、10,000g、60分の条件で遠心分離し、フィルター下部に移行した濾液をそのまま質量分析用の試料とした。
従来の方法による血清のトリプシン消化ペプチドの調製工程を以下に説明する。1.5mLエッペンドルフチューブに粉末ウレア10mgを量りとり、上述のように調製した血清10μLを添加した(変性)。混合後、50mmol/Lのトリス(2−カルボキシエチル)ホスフィン塩酸塩を2μL加え、37℃,30分間の条件で反応させた(還元)。次に、0.5mol/Lのヨードアセトアミド及び1mol/Lの重炭酸アンモニウムの混合溶液を0.8μL加え、常温で45分間反応させた(アルキル化)。これを160μLの10mmol/Lのトリス塩酸緩衝液(pH8.0)で希釈し、さらに前述のトリプシン溶液を2μL加えて、37℃で16時間反応させた(血清試料10μLに対してトリプシン約20μg)。反応後のペプチド溶液に10%トリフルオロ酢酸を4μL加え、質量分析用の試料とした。
上記で得られた試料0.5μLを、ZipTip(商標、ミリポア社)により脱塩した。この際、試料を1nmol/mLの標準ペプチド(アミノ酸配列PPPPPPPPPPPPPPR(配列番号12)、以下P14R)を含む10μLの0.1%トリフルオロ酢酸にて希釈し、溶出液の1/10をμFocusingプレート(島津GLC)にスポッティングした。風乾後、マトリックスとして3mg/mLの2,5−ジヒドロキシ安息香酸を1μLスポッティングし、結晶化の後、MALDI MS(AXIMA−Resonance、島津製作所)にて測定した。Positive MidMassモード、レーザー強度100の条件でのラスター駆動により200箇所から合計200プロファイルを取得し積算することで、各スポットよりマススペクトルを取得した。
MS/MSの測定は、対象のモノアイソトピックマスに対してイオントラップを設定したうえで、Positive Midmassモード、レーザー強度110の条件でのラスター駆動により200箇所から合計200プロファイルを取得し、積算した。CID(Collision Induced Dissociation、衝突誘起解離)の強度設定値は対象にあわせて適宜調整した。
マススペクトルからP14Rに由来するシグナル(m/z 1533.9)の強度及びANXA4(配列番号7のアミノ酸配列を有するペプチド)に由来するシグナル(m/z 1379.6)の強度をそれぞれ抽出し、集計した。また、MS/MSモードにより取得したm/z 1379.6のプロダクトイオンスペクトルより、プロダクトイオンm/z 458.3の強度を抽出し、集計した。MSモードにおける信号強度はm/z 1533(P14R)に対してノーマライズを行い、イオン化に伴う信号強度の強弱について補正を行った。
本発明による前処理方法及び従来法による前処理により得られた血清消化ペプチドのマススペクトルを図1に示す。観測されるピークのうち、アルブミンに由来するピークを*印により記した。比較すると、従来法(上段)においてはスペクトル中に観測される主要なピークのほぼ全てがアルブミンに由来するのに対して、本発明による方法(下段)ではアルブミン由来のピークの発生が抑えられていることが示された。
[分析対象ペプチドの選定]
本発明の方法に好適なペプチドの候補として上記に挙げた配列番号2−11のペプチドがあるが、この中から特に好適なペプチドを選択するため、α2マクログロブリン(A2M)を用いた予備実験を実施した。
[試料及びプロテアーゼの調製]
実施例1と同様にして行った。
トリプシンによる消化条件を4℃で16時間とする以外は実施例1と同様にして行った。
実施例1と同様にして行った。
上記で得られた試料0.5μLを、ZipTipにより脱塩し、溶出液に10pmolの標準ペプチドP14Rを添加し、その1/10量をμFocusingプレート(島津GLC)にスポッティングした。風乾後、マトリックスとして3mg/mLの2,5−ジヒドロキシ安息香酸を1μLスポッティングし、結晶化の後、MALDI MS(AXIMA−Resonance、島津製作所)にて測定した。Positive MidMassモード、レーザー強度100の条件でのラスター駆動により200箇所から合計200プロファイルを取得し積算することで、各スポットよりマススペクトルを取得した。
実施例1と同様にして行った。
マススペクトルから信号強度0.5mVを閾値にピーク検出を行い、その中からP14Rに由来するシグナル(m/z 1533.9)の強度及びANXA4(配列番号2のアミノ酸配列を有するペプチド)に由来するシグナル(m/z 1581.7)の強度をそれぞれ抽出し、集計した。また、MS/MSモードにより取得したm/z 1581.7のプロダクトイオンスペクトルより、プロダクトイオンm/z 588.3(配列番号2のy5イオンに相当)の強度を抽出し、集計した。この際、閾値0.5mVのピーク検出処理において認識されなかったシグナルを不検出(ND)とした。MSモードにおける信号強度はm/z 1533(P14R)に対してノーマライズを行い、イオン化に伴う信号強度の強弱について補正を行った。
図5は、m/z 1581.7のピーク周辺の拡大図である。矢印で示すように、従来法(A)による前処理では検出されないこのピークは、本発明による前処理(B)により38μg/ml以上で検出された。この結果から明らかなように、従来の方法ではANXA4由来のピークは全く検出されなかったのに対し、本発明の方法では濃度に依存したピークが観察された。なお、本実験条件において、ANXA4由来ペプチドの候補ペプチド(配列番号2−11)のうち、マススペクトル中から実際に検出されたのは配列番号2及び3のみであり、実施例2において好適とされたペプチドと一致していることが確認された。
Claims (9)
- 血清又は血漿試料中のタンパク質を質量分析によって検出するための試料の前処理方法であって、タンパク質の未変性条件下で該試料にプロテアーゼを添加して消化する工程と、得られたペプチドを未消化のタンパク質と分離する工程とを含む、上記前処理方法。
- 血清又は血漿試料中のタンパク質を質量分析によって検出するための方法であって、タンパク質の未変性条件下で該試料にプロテアーゼを添加して消化する工程と、得られたペプチドを未消化のタンパク質と分離する工程と、該ペプチドを質量分析に供する工程とを含む、上記方法。
- プロテアーゼ処理の前及び/又は後にタンパク質又はペプチドの濃縮工程を含まない、請求項1又は2記載の方法。
- プロテアーゼがトリプシンである、請求項1〜3のいずれか1項記載の方法。
- プロテアーゼとして固定化酵素を用いる、請求項1〜4のいずれか1項記載の方法。
- 試料中にα2マクログロブリンが存在する、請求項1〜5のいずれか1項記載の方法。
- LC/MS又はMALDI−TOF法によって分析する、請求項1〜6のいずれか1項記載の方法。
- MS/MS分析を行う、請求項1〜7のいずれか1項記載の方法。
- アルブミンが消化されない、請求項1〜8のいずれか1項記載の方法。
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