Method for processing samples for peptide mapping analysis

JP7872263B2Active Publication Date: 2026-06-09AMGEN INC
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Patent Information

Authority / Receiving Office
JP · JP
Patent Type
Patents
Current Assignee / Owner
AMGEN INC
Filing Date
2021-09-17
Publication Date
2026-06-09

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【0013】 本開示は、ポリペプチドを処理して、各々がC末端にチロシンを含む少なくとも2種のペプチドを含む消化試料を生成する方法をさらに提供する。例示的な実施形態において、本方法は、約1:1~約1:15のE:S比でポリペプチドをトリプシンにより消化することを含む。様々な例において、消化は、本明細書に開示されている方法に従って行われる。任意選択で、消化試料は、HGNFGNSY(配列番号108)のアミノ酸配列を含むペプチド、HGNFGNSYISY(配列番号109)のアミノ酸配列を含むペプチド、及び/又はHGNFGNSYISYWAY(配列番号110)のアミノ酸配列を含むペプチドを含む。例示的な態様において、E:S比は、約1:1~約1:10である。任意選択で、E:S比は、約1:2~約1:8である。様々な例において、E:S比は、約1:4~約1:6、任意選択で、約1:5である。例示的な態様において、消化は、約7.0~約8.0、任意選択で約7.5のpHで行われる。様々な態様において、消化は、約12時間未満、約6時間未満、約4時間未満行われる。様々な例において、消化は、約2時間から約4時間行われる。様々な態様において、消化は、約2時間以下行われる。例示的な例において、消化は、約35℃~約40℃、任意選択で、約37℃の温度で行われる。任意選択で、消化は、塩化カルシウムの存在下で行われる。或いは、消化は、塩化カルシウムの不在下で行われる。例示的な例において、ポリペプチドは、約1:1~約1:15のE:S比でトリプシンのみにより消化される。様々な態様において、他のプロテアーゼは、ポリペプチドを消化するために使用されない。様々な態様において、ポリペプチドをこのE:S比でトリプシンにより消化すると、C末端にチロシンを含む1種以上のペプチドを含む消化試料が生成される。例示的な態様において、本方法は、ポリペプチドをプロテアーゼにより消化する前に、ポリペプチドを変性させること、ポリペプチドを還元すること、及び/又はポリペプチドをアルキル化することを含む。例示的な例において、本方法は、ポリペプチドをプロテアーゼにより消化する前、且つポリペプチドを変性、還元、及び/又はアルキル化した後に、バッファ交換を含み、任意選択で、バッファ交換は、サイズ排除カートリッジの使用を含む。例示的な態様において、サイズ排除カートリッジは、Sephadex G-25ゲルろ過材料を有するNAP5カートリッジである。例示的な態様において、本方法は、消化試料を弱酸性のpHで、カオトロープの存在下でインキュベートすることをさらに含む。任意選択で、カオトロープは、塩酸グアニジンである。弱酸性のpHは、様々な態様において約5である。様々な例における方法は、消化試料のペプチドを、ペプチドマッピング分析のために液体クロマトグラフィー質量分光測定(LC-MS)システムに注入することを含む。

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Abstract

Provided herein are methods for processing polypeptides or proteins for analysis, e.g., peptide mapping analysis by mass spectrometry. In exemplary embodiments, the methods include incubating a digested sample at a mildly acidic pH and / or in the presence of a chaotrope, wherein the digested sample is generated by digesting the polypeptide with a protease to generate a digested sample containing at least two peptides. In exemplary embodiments, the methods include digesting the polypeptide with a protease that cleaves the C-terminus of tryptophan to generate a digested sample containing at least two peptides. In exemplary embodiments, the methods include digesting with trypsin at an enzyme:substrate (E:S) weight ratio of about 1:1 to about 1:15 to generate a digested sample containing at least two peptides. In exemplary aspects, the digested sample contains at least one or two peptides, each containing a tyrosine at its C-terminus.
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Claims

1. A method for processing a polypeptide to produce a digested sample containing at least one or two peptides, each having tyrosine at its C-terminus, comprising digesting the polypeptide with trypsin in an enzyme:substrate (E:S) weight ratio of 1:2 to 1:10 to obtain a digested sample containing one or two peptides, each having tyrosine at its C-terminus.

2. The method according to claim 1, wherein the E:S ratio is 1:4 to 1:6, and optionally 1:

5.

3. The method according to claim 1 or 2, wherein the digestion is carried out at a pH of 7.0 to 8.0, optionally 7.

5.

4. The method according to any one of claims 1 to 3, wherein the digestion is carried out for less than 12 hours, or optionally less than 6 hours.

5. The method according to claim 4, wherein the digestion is performed for less than 4 hours, or optionally for 2 to 4 hours.

6. The method according to any one of claims 1 to 5, wherein the digestion is carried out at a temperature of 35°C to 40°C, optionally 37°C.

7. The method according to any one of claims 1 to 6, wherein the digestion is carried out in the presence of calcium chloride.

8. The method according to any one of claims 1 to 6, wherein the digestion is carried out in the absence of calcium chloride.

9. The method according to any one of claims 1 to 8, wherein only trypsin is used to digest the polypeptide.

10. The method according to any one of claims 1 to 9, wherein the digested sample comprises a peptide containing the amino acid sequence of HGNFGNSY (SEQ ID NO: 108), a peptide containing the amino acid sequence of HGNFGNSYISY (SEQ ID NO: 109), and / or a peptide containing the amino acid sequence of HGNFGNSYISYWAY (SEQ ID NO: 110).

11. The method according to any one of claims 1 to 10, comprising denaturing the polypeptide, reducing the polypeptide, and / or alkylating the polypeptide before digesting the polypeptide with the trypsin.

12. The method according to any one of claims 1 to 11, comprising buffer exchange before digesting the polypeptide with the trypsin and after denaturing, reducing, and / or alkylating the polypeptide.

13. The method according to claim 12, wherein the buffer replacement is performed by gel filtration using a size exclusion cartridge.

14. The method according to any one of claims 1 to 13, further comprising incubating the digested sample at a weakly acidic pH in the presence of chaotrope.

15. The method according to claim 14, wherein the chaotrope is guanidine hydrochloride.

16. The method according to claim 14 or 15, wherein the pH of the weakly acidic area is 5.

17. The method according to any one of claims 1 to 16, comprising injecting the peptide of the digested sample into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis.

18. A method for monitoring polypeptide attributes, a. Processing the first polypeptide in the first sample obtained at a first time point according to the method described in any one of claims 1 to 17, b. The peptides from the digested sample are injected into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis, and the PTM of the polypeptide in the first sample is identified. c. Processing a second polypeptide in a second sample obtained at a second time point according to the method of any one of claims 1 to 17, wherein the second polypeptide is the same as or different from the first polypeptide. d. The peptides from the digested sample are injected into a liquid chromatography-mass spectroscopy (LC-MS) system for peptide mapping analysis, and the PTM of the polypeptide in the second sample is identified. e. Comparing the PTM of the first sample with the PTM of the second sample. A method that includes this.