JP5902357B2 - 質量分析によるサイログロブリン定量 - Google Patents
質量分析によるサイログロブリン定量 Download PDFInfo
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- JP5902357B2 JP5902357B2 JP2015533192A JP2015533192A JP5902357B2 JP 5902357 B2 JP5902357 B2 JP 5902357B2 JP 2015533192 A JP2015533192 A JP 2015533192A JP 2015533192 A JP2015533192 A JP 2015533192A JP 5902357 B2 JP5902357 B2 JP 5902357B2
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- peptide
- ions
- thyroglobulin
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/046—Thyroid disorders
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7023—(Hyper)proliferation
- G01N2800/7028—Cancer
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
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- Pathology (AREA)
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- Organic Chemistry (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本出願は、米国特許法第119条(e)の下で、ここに本開示の一部を構成するものとしてその内容全体を援用する、2012年9月20日に出願された米国特許仮出願第61/703,721号に基づく利益を主張するものである。
試料を加工または精製して、質量分析による解析に適した調製物を得ることができる。斯かる精製は通常、液体クロマトグラフィー等のクロマトグラフィーを含み、多くの場合、クロマトグラフィーの前に行われる追加の精製手順を含んでいてもよい。試料の種類またはクロマトグラフィーの種類に応じて、様々な手順をこの目的のために用いることができる。例として、濾過、遠心分離、これらの組み合わせその他が挙げられる。特定の好ましい実施形態において、Tgは、酵素消化の前に被験試料に存在する。
様々な実施形態において、Tgペプチドは、当業者に公知のいずれかの方法によりイオン化することができる。質量分析は、分画された試料をイオン化し、さらなる解析のための荷電分子を作製するためのイオン源を含む質量分析計を用いて行われる。様々なMS技術において用いられるイオン化源として、電子イオン化、化学イオン化、エレクトロスプレーイオン化(ESI)、光子イオン化、大気圧化学イオン化(APCI)、光イオン化、大気圧光イオン化(APPI)、高速原子衝撃(FAB)/液体二次イオン化(LSIMS)、マトリックス支援レーザー脱離イオン化(MALDI)、電界イオン化、電界脱離、熱スプレー/プラズマスプレーイオン化、表面増強レーザー脱離イオン化(SELDI)、誘導結合型プラズマ(ICP)および粒子ビームイオン化が挙げられるがこれらに限定されない。当業者であれば、イオン化方法の選択が、測定しようとする分析物、試料の種類、検出器の種類、正又は負のモードの選択等に基づき決定され得ることを理解できよう。
サイログロブリンの不完全な消化は、サイログロブリン定量の不正確性の原因となり得る。いくつかの実施形態において、サイログロブリンペプチド標準を、消化の前に被験試料に添加することができる。サイログロブリンペプチド標準は、いくつかの実施形態において、消化されると、サイログロブリンにより産生されるペプチドと同じ1種または複数のTgペプチドを産生する。一部の態様において、試料に添加されるサイログロブリンペプチド標準の量は公知である。
公知のペプチドT129濃度の試料を初発とする連続希釈により、様々な公知の濃度のペプチドT129を含む数種類の試料を調製した。これらの試料のLC−MS/MS解析から、ペプチドT129 LOQおよび較正曲線を作成した。
ストリップ血清(例えば、本実施例における被験試料)の500μl試料を、市販の300kDa分子量カットオフフィルターカートリッジ(Pall Corp.Nanosep 300kDa、Pall Corp.カタログ番号OD300C33)のフィルター要素の上に加えた。
実施例2に詳述されている手順に従って、様々な濃度の添加Tgを含有するストリップ血清の500μl試料を数種類調製した。実施例1に詳述されているステップに従って、その結果得られた被験試料のLC−MS/MSを行った。
Tgが十分に消化されていることを確認するために、消化されると同位体標識T−129内部標準を遊離する、合成「翼状(winged)」ペプチドを被験試料に加えた。理想的には、調製プロセス(変性/還元、アルキル化および消化)の前に血清試料に加えることのできる、完全に標識されたTgが用いられる。しかし、現在、斯かる標識Tgタンパク質を得ることは不可能である。従って、その中に同位体標識されたT−129を含有する短いTgペプチドを合成した(T129−IS1)。T129−IS1は、血清試料に存在するTg由来のT129と同じ様式で、加工手順において生成される。従って、これは、標識Tgタンパク質の代用として作用し、試料の完全な加工を確認する。
イムノアッセイおよび本質量分析方法の両方を用いて、患者試料中のTgを定量した。次表に示す通り、検査から得られた結果は、抗体陰性試料に対して十分に相関したが、患者被験試料が抗体陽性である場合、非常に異なる結果を産生した。下表は、有意なTgAb濃度の存在下において、イムノアッセイにより検出されるTgの量は低いが、一方、LC−MS/MSにより決定されるTgの量はより高いことを示す。
本実施例において、DTCを呈する37歳女性は、根治的甲状腺摘除術および放射性ヨウ素によるアブレーションを受けた。この患者は、Tg抗体(Ab)陽性であることが判明した。自動ICMAプラットフォームにおいて患者を検査したところ、結果は0.2ng/mL Tgであった。RIA検査のために試料を送ったところ、15ng/mLのTgであり、ICMA検査とは矛盾した結果であった。本技術のLC−MS/MSアッセイを行った。後者のアッセイは、干渉抗体を破壊し、5.6ng/mL Tgの結果を返し、患者が経過観察を要し、さらなる外科手術を必要とする可能性があることを示唆する。
Claims (13)
- 被験試料中のサイログロブリンの量を決定するための方法であって、
(a)前記被験試料中のサイログロブリンおよび添加した同位体標識サイログロブリンペプチド標準を消化して、TgペプチドおよびTgペプチド標準産物を生成するステップと、ここで、Tgペプチドはアミノ酸配列VIFDANAPVAVR(配列番号1)を含むとともに、Tgペプチド標準産物は1またはそれ以上のバリンが13C、15Nにより同位体標識されており、
(b)ステップ(a)からの前記TgペプチドおよびTgペプチド標準産物を精製するステップと、
(c)ステップ(b)からの前記TgペプチドおよびTgペプチド標準産物をイオン化して、質量分析によって検出可能な1種または複数のTgペプチドイオンおよびTgペプチド標準産物イオンを産生するステップと、
(d)質量分析によって、ステップ(c)からのイオンの量を検出するステップであって、ステップ(d)で検出されるイオンの量を、ステップ(a)で消化された前記被験試料中のサイログロブリンの量およびサイログロブリンペプチド標準の量と関係付けるステップと、
を含む方法。 - サイログロブリンペプチド標準の長さが、50アミノ酸残基未満である、請求項1に記載の方法。
- TgペプチドおよびTgペプチド標準産物の両方が、T129ペプチド(配列番号1、VIFDANAPVAVR)を含む、請求項2に記載の方法。
- サイログロブリンペプチド標準が、配列番号2のアミノ酸配列(KVPESKVIFDANAPVAVRSKVPDS)を含む、請求項3に記載の方法。
- サイログロブリンペプチド標準が、配列番号2(KVPESKVIFDANAPVAVRSKVPDS)に対し少なくとも85%の配列同一性を有するアミノ酸配列を含み、ステップ(a)の消化によりT129ペプチド(配列番号1、VIFDANAPVAVR)を生成することができる、請求項1に記載の方法。
- 被験試料に添加された同位体標識サイログロブリンペプチド標準の量が既知である、請求項1に記載の方法。
- ステップ(c)において産生されたTgペプチドイオンが、541.3±0.5、612.3±0.5、636.4±0.5、726.4±0.5、797.4±0.5、912.4±0.5または1059.5±0.5の質量/電荷比を有するイオンの群から選択される1種または複数のイオンを含む、請求項1に記載の方法。
- 前記イオン化が、636.4±0.5の質量/電荷比を有するTgペプチド前駆イオンを生成することと、797.4±0.5、912.4±0.5または1059.5±0.5の質量/電荷比を有する1種または複数の断片イオンを生成することとを含む、請求項1に記載の方法。
- 前記被験試料が、体液または組織である、請求項1に記載の方法。
- 被験試料中のサイログロブリンの量を決定するための方法であって、
(a)前記被験試料中のサイログロブリンを消化して、ペプチドT129を生成し、前記被験試料中の添加された同位体標識サイログロブリンペプチド標準を消化して、同位体標識ペプチドT129内部標準を生成するステップと、ここで、ペプチドT129はアミノ酸配列VIFDANAPVAVR(配列番号1)を含むとともに、同位体標識ペプチドT129内部標準は1またはそれ以上のバリンが13C、15Nにより同位体標識されており、
(b)ステップ(a)からの前記ペプチドT129およびペプチドT129内部標準を精製するステップと、
(c)ステップ(b)からの前記ペプチドT129およびペプチドT129内部標準をイオン化して、タンデム質量分析によって検出可能な2種以上の前駆イオンを生成するステップであり、ペプチドT129の前記前駆イオンが、636.4±0.5の質量/電荷比を有するステップと、
(d)質量分析装置において前記前駆イオンを断片化して、質量分析によって検出可能な1種または複数の断片イオンを生成するステップであって、ペプチドT129の前記断片イオンのうち1種または複数が、797.4±0.5、912.4±0.5または1059.5±0.5の質量/電荷比を有するイオンのリストから選択されるステップと、
(f)質量分析により、ステップ(d)の前記前駆イオン、ステップ(e)の前記断片イオンのうち1種もしくは複数、またはその両方の量を検出するステップと
を含み、ステップ(f)で検出されたイオンの量を、前記被験試料中の前記サイログロブリンの量と関係付ける、方法。 - サイログロブリンペプチド標準が、配列番号2のアミノ酸配列を含む、請求項10に記載の方法。
- サイログロブリンペプチド標準が、配列番号2(KVPESKVIFDANAPVAVRSKVPDS)に対し少なくとも85%の配列同一性を有するアミノ酸配列を含み、ステップ(a)の消化によりT129ペプチド(配列番号1、VIFDANAPVAVR)を生成することができる、請求項10に記載の方法。
- 被験試料に添加された同位体標識サイログロブリンペプチド標準の量が既知である、請求項10に記載の方法。
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