WO2016028075A1 - 마이크로 중공사막 효소 반응기 - 텐덤 질량분석법을 이용한 단세포군 항체 기반 온라인 인단백질 프로테오믹스 분석방법 - Google Patents

마이크로 중공사막 효소 반응기 - 텐덤 질량분석법을 이용한 단세포군 항체 기반 온라인 인단백질 프로테오믹스 분석방법 Download PDF

Info

Publication number
WO2016028075A1
WO2016028075A1 PCT/KR2015/008641 KR2015008641W WO2016028075A1 WO 2016028075 A1 WO2016028075 A1 WO 2016028075A1 KR 2015008641 W KR2015008641 W KR 2015008641W WO 2016028075 A1 WO2016028075 A1 WO 2016028075A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
mass spectrometry
extraction
antibodies
peptide
Prior art date
Application number
PCT/KR2015/008641
Other languages
English (en)
French (fr)
Inventor
강덕진
이선영
김숙경
Original Assignee
한국표준과학연구원
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 한국표준과학연구원 filed Critical 한국표준과학연구원
Priority to US15/504,732 priority Critical patent/US20170261513A1/en
Publication of WO2016028075A1 publication Critical patent/WO2016028075A1/ko

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • G01N27/622—Ion mobility spectrometry
    • G01N27/623—Ion mobility spectrometry combined with mass spectrometry
    • 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
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/0027—Methods for using particle spectrometers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/004—Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn

Definitions

  • the present invention relates to serine-phosphate, threonine-phosphate and tyrosine-phosphate specific affinity in post-translational modifications (PTMs) of proteins.
  • PTMs post-translational modifications
  • MHFER-based on-line peptide-specific pretreatment using antibodies with antibodies, and the peptides recovered through the on-line pre-treatment can be analyzed using a tandem mass spectrometer for the qualitative analysis of oxidized proteins and their phosphorylation sites in proteins. It is about analytical techniques that can be identified.
  • Qualitative and quantitative analysis of proteins in samples is used to diagnose human diseases.
  • protein PTMs are chemical modifications that interact with other cellular molecules such as proteins, nucleic acids, lipids, and cofactors. Involved in the regulation of activity, it plays a key role in the functional aspects of the protein.
  • phosphoproteins are known to affect intercellular interactions, cell death and production, and on / off of protein function.
  • Selective pretreatments include hydrophilic interaction liquid chromatography (HILIC), strong anion-exchange chromatography (SAX), and strong cationic ion-e.
  • Peptides are separated using xchange chromatography (SCX), and then positive charges of metal ions and After pretreatment such as iminobilized metal affinity chromatography (MAC) and metal oxide affinity chromatography (MOAC) using chemical bonds between negative charges, only phosphoproteins are extracted and then introduced into a mass spectrometer.
  • Electrophoresis (2D gel electrophoresis, 2D GE), immunooblot and immunoprecipitation (IP) are used.
  • Antigen-specific binding proteins can be identified in gels by using the binding principle of Antigen, and this method has the advantage of extracting only the proteins to be analyzed effectively. Not all antibodies are applied to the silanol group of the membrane or beads so that unwanted proteins are extracted and reproduced Is lowered, the other pre-treatment such as handling the albumin to silanol antibody is not coated is necessary to prevent this. In addition, when the extracted protein is introduced into a mass spectrometer, a second pretreatment process (removal of the surfactant used during the extraction or the step of peptidetizing the protein) requires a sample loss due to complex pretreatment.
  • the low-efficiency of the extraction method using the affinity between the in-peptide or the phosphorus protein and the metal ion and the on-line antibody-specific antibody using the micro hollow fiber membrane enzyme antifungal-based antigen antibody reaction can improve the complex sample preparation process. It was intended to provide a body mass spectrometry.
  • the present invention comprises the steps of: a) adding a reducing agent to a protein complex present in the cell to denature it, and then reacting with an enzyme to obtain a peptide complex; b) combining the peptide mixture of step a) with a phosphoprotein or an peptide specific antibody; and c) injecting the reaction product obtained in step b) into a micro hollow fiber membrane enzyme reaction group (mHFER) and enzymatically treating it.
  • MHFER micro hollow fiber membrane enzyme reaction group
  • the protein complex used in step a) is not limited, but cell lysates extracted from cells may be used.
  • the reducing agent is not limited, but dithiothreitol (DTT) is used. Dithioerythritol, tris 2-carboxyethyl phosphine or tributyl phosphine may be used.
  • the protein may be denatured using dithiotray (DTT).
  • the enzyme is not limited, but may be used as a protease, and preferably trypsin.
  • the thiol group (-SH) of cysteine is iodoacetamide (IAA) to prevent refolding of disulfide bonds of cysteine-cysteine. It is transformed into an irreversible form by the addition of alkylation. Residual IAA can lead to light deformation or additional reaction, thus removing L-cysteine.
  • the weight ratio of the solution between trypsin and protein is 1:40 to 1
  • the enzyme can be added by adding 60: reaction in a temperature stirrer for 18 hours at 37 ° C.
  • the peptide or phosphorus protein-specific antibody is not limited, but is composed of phosphoserine-, intreonine- and intyrosine-antibodies having a molecular weight of 50 kDa or more. Either one or more than one antibody combination may be used.
  • the erythro-antibody may be serine-phosphate and the intreonine-antibody may be threonine-phosphate and intiro.
  • the neo-antibody has affinity with tyrosine-phosphate, respectively.
  • the mixing ratio of the peptide or the phosphoprotein with the antibody is not limited, the phosphorus protein or the phosphoprotein with respect to 100 parts by weight of the antibody or antibody complex is not limited. The mixture may be 10 to 1000 parts by weight, and the reaction forms a bond between the serine-, threonine- and tyrosine-phosphate group and each antibody.
  • the hollow fiber membrane used in step c) is not limited, but preferably
  • Molecular weight transmission limit is 10 kDa, inner diameter is about 200 to 600 ⁇ , outer diameter is about 500 to
  • Polyvinyl chloride, polyacrylonitrile or combinations thereof are preferred.
  • the extraction may be performed in the range of 4 to 25 ° C, although not limited thereto, and may be carried out within the range according to one embodiment of the present invention.
  • the antibody-binding peptide mixture obtained from b) is injected into the hollow fiber membrane enzyme reactor (mHFER), the antibody-binding peptide having a size of 10 kDa or more stays in mHFER and does not bind to the antibody. Peptides less than 10 kDa in molecular weight are released through mHFER and eluted.
  • the reaction results in the degradation of the antibody by injecting a protease such as trypsin, thereby losing the function of the peptide, which can elute the peptide bound to the antibody.
  • the peptide is trapped in a reverse trapping column.
  • a reverse C18 column which is a separation means according to the degree of hydrophobicity, it is introduced into a mass spectrometer through electrospray ionization (ESI).
  • the mass spectrometer of step c) is not limited, for example, Flow-flow liquid chromatography (nLC, 1260 capillary LC system, Agilent Technologies, Germany) -electrospray ionization-Fourier transform orbitlab-tandem mass spectrometry (ESI-FT orbitrap-MS / MS, Q-Exactive, Thermo Scientific, Germany) Can be used
  • the online peptide or phosphoprotein extraction method according to the present invention detects antibodies having a relatively low concentration of phosphorylated temide specific affinity.
  • the automated system configuration using the mHFER device has the advantage of minimizing the problems associated with existing complex pretreatments. .
  • FIG. 1 shows that after enzymatic treatment of the protein obtained using a cell sample (MCF7),
  • Figure 3 shows the antibody-binding peptide or phosphoprotein extraction for 10 ⁇ g antibody
  • FIG. 4 shows the interaction between in-peptide or phosphoprotein and antibody in the case of FIG.
  • Figure 5 shows serine-antibody, intreonine-antibody and
  • Figure 6 shows the number of in peptides extracted by the extraction method and the existing phosphoprotein extraction method (FASP, IMAC, Ti0 2 ) developed in the peptide complex (10 ⁇ ⁇ ) obtained from MCF7 cell lysate.
  • FIG. 7 shows the number of phosphorylation sites present in serine, threonine and tyrosine in the extracted phosphorous peptides as a result obtained from FIG. 6.
  • Test Example 1 Produced from proteins present in cell lysates
  • MCF7 Kerme Cell Line Bank, Republic of Korea cells (5 * 10 6/10 cm dish) for collecting and into a (phosphate buffered saline), 0.1 M PBS and then ultrasonic grinding using a tip sonicator, 10 minutes at 10,000 rpm The supernatant was separated, 100 ⁇ ⁇ of the extracted protein was mixed with a 50 mM ammonium bicarbonate solution and a solution containing 10 mM DTT, and then denatured at 37 ° C. for 2 hours. 32 ⁇ L of 270 mM IAA solution was added to the solution and alkylated in a room temperature dark room for 30 minutes. 47 ⁇ L of L-cysteine was added to remove residual ⁇ . Subsequently, 2 [xg trypsin was added and peptideized at 37 ° C. for 18 hours.
  • Antibody-binding peptide mixtures were prepared by incorporating inserine-antibodies, intreonin-antibodies and intyrosine-antibody mixtures in the composition and temperature conditions of Table 1 below.
  • the permeation limit is 10 kDa and is about 5 volumes.
  • the reaction caused the degradation of the antibody, causing the peptides that had bound to the antibody to fall off and eluted. It is connected to the flow path of the MS instrument, and the C18 is connected to the reverse solvent by the binary pump.
  • the layered column was eluted according to the degree of hydrophobicity of the peptide and introduced into the mass spectrometer.
  • Example 1 to Example 3 relates to the number of extracted phosphoproteins and phosphopeptides according to the weight ratio between the protein or the peptide and the antibody, the weight ratio between the peptide and the antibody according to the result of FIG. 3 is 1:10, 1 At 1: 1 and 10: 1, the number of measured peptides was 400, 553, and 229, respectively. This resulted in the highest number of extracted peptides when the weight ratio was 1: 1. .
  • reaction temperature was 25 ° C, 553 peptides were identified, and when the reaction temperature was 25 ° C, 296 peptides were identified. Therefore, when the reaction temperature was 25 ° C, the number of extracted peptides increased. It was.
  • Example 5 to Example 7 were compared by comparing the protein or peptide with each antibody (in serine-antibody, intreonine-antibody and intirosine-antibody using two antibodies of different sources). As shown in FIG. 5, the number of in peptides measured before extraction was 272 and measured using inserine-antibodies I and ⁇ .
  • the number of inpeptides was 1,438 and 1,229. Inteonin-antibodies I and II were measured to be 1,397 and 202. The number of inpeptides extracted by intirosine-antibodies I and II was 713 and 76. It was confirmed as an individual, which was measured when each antibody was used as compared with that which was not extracted based on the results.
  • Ni-NTA spin column Qiagen, Hilden, Germany
  • the Ni-NTA beads were sol-gel frits of 3 mm porous glass material at one end. It was used by layering in a clogged capiUary (inner diameter 200um, length 100mm) in order of syringe pump, sample injector and capillary layered with Ni-NTA beads, and then pouring the solvent at a flow rate of 1 to 5 ⁇ ! 7 ⁇ . It is set up so that can be introduced.
  • NTA beads were equilibrated by pouring loading buffer (0.1 M NaOH solution with 6% acetic acid, pH 3.6) into the capillary for 30 minutes. A 10 peptide mixture was injected through the sample injector. Thereafter, the phosphate protein or phosphopeptides were chelated to Fe-NTA beads by 20 minutes of loading buffer.
  • loading buffer 0.1 M NaOH solution with 6% acetic acid, pH 3.6
  • wasing buffer (loading buffer / acetontrile, 75/25, v / v) was applied to the capillary to remove unchelated peptides from the capillary. Prior to this, 100 loading buffers were loaded to equilibrate, and 4% NH 4 OH solution at pH 11 was collected to elute the chelated peptides. The collected solution was collected using a vacuum concentrator. After drying, the solution was re-dissolved in a 0.1% formic acid solution and introduced into a mass spectrometer.
  • SPE Solid phase extration
  • a 10 Hg peptide mixture was placed in a Ti0 2 cartridge, 1000 buffer B per 500 samples were mixed in the cartridge, and centrifuged at 200 xg for 5 minutes. The collected solution was then placed in a Ti0 2 cartridge and centrifuged under the same conditions. The procedure was repeated 10 times to allow more phosphorus peptides to chelate on Ti0 2 beads.
  • 200 nL of buffer B was centrifuged at 200 xg for 2 minutes, and 200 A of buffer A was washed at 200 xg for 2 minutes.
  • FIG. 7 shows the number of phosphorylation present in the serine, threonine and tyrosine positions in the in-peptide through the experiment of FIG. 6.
  • the present invention was completed by confirming that not only single peptides but also multifactor peptides including double and triple peptides which were not known in the past when the antibody complex extraction method was used.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

본 발명은 마이크로 중공사막 효소반응기 (microbore hollow fiber enzymatic reactor, tnHFER)기반 항원항체반응을 이용한 인단백체 추출법 및 질량분석 방법에 관한 것으로, 상세하게는 체내 존재하는 인단백질 또는 인펩타이드를 인세린-，인트레오닌-，및 인티로신-항체를 이용하여 추출하여 질량분석기로 측정함으로써, 질환의 진단을 위한 바이오마커 인단백질을 발굴하고，이를 통하여 질환의 조기진단에 기여할 수 있는 추출방법 및 질량분석법에 관한 것이다. 본 발명의 항원항체반응 기반 추출법을 이용한 질량분석법은 낮은 추출율 및 복잡한 시료전처리를 통한 방법에서 발생하는 시간적 ㅇ경제적 부담을 최소화할 수 있으며, 상당수의 인펩타이드 (또는 인단백질)과 친화력이 강한 항체를 사용함으로써 추출효율을 높이며, 저농도의 인펩타이드 또는 인단백질 추출이 가능해짐에 따라 질병의 진단 단백질 마커 발굴 및 메커니즘 규명 연구에의 활용 가능성이 높을 것으로 기대된다.

Description

명세서
발명의명칭:마이크로중공사막효소반웅기 -텐덤질량분석법을 이용한단세포군항체기반온라인인단백질프로테오믹스 분석방법
기술분야
[1] 본발명은단백질의전사후수식들 (post-translational modifications, PTMs)중 세린-인산기 (serine-phosphate),트레오닌-인산기 (threonine-phosphate), 티로신-인산기 (tyrosine-phosphate)특이적친화력을갖고있는항체들을이용한 mHFER기반온라인인펩타이드특이적전처리법개발에관한것이며，상기 온라인전처리과정을통해회수된인펩타이드들은텐덤질량분석기를 이용하여생체시료중인산화단백질의정성분석및단백질내인산화위치까지 확인할수있는분석기술에관한것이다.
배경기술
[2] 질량분석기를기반으로한단백체학 (proteomics)연구는단백질의구조규명및 정량분석에서중요한역할을하고있으며,유전자의기능을이해하기위한 수단으로써사용되고있다.사람으로부터얻을수있는다양하고복잡한 생체시료내에존재하는단백질을정성및정량분석함으로써，인간의질병을 진단하는목적으로이용되고있다.일반적으로단백질 PTMs은화학적 변형으로써단백질，핵산,지질그리고보조인자와같은다른세포분자들간의 상호작용및활성조절에관여하므로,단백체의기능적인측면에서핵심적인 역할을하고있다.특히,인단백질 (Phosphoprotein)은세포간의상호작용，세포의 사멸과생성및단백질기능의 on/off에영향을준다고알려져있다.또한， 대부분의인산화는단백질의아미노산배열중세린 (serine)에많이분포하고 있으며，트레오닌 (threonine)과타이로신 (tyrosine)에도인산화가이루어진다고 알려져있다.인단백질은인체내존재하는다른단백질들에비해상대적으로 적은양으로존재하며,특히，인산기가가지는음전하로기인하여양이온 모드 (positive ion mode)기반전기분무이온화 -텐덤질량분석기 (electrospray ionization-tandem mass spectrometer, ESI-MS/MS)를이용한프로테오믹스분석 시，일반적인효소처리과정을통해생성된펩타이드들에비해상대적으로낮은 ESI효율을갖으며，결과적으로인펩타이드들에대한정성및정량분석에 어려움을갖고있다.상기이유로기존인단백질체또는인펩타이드들에대한 질량분석기기반정성분석효율을높이기위해다양한전처리방법들이개발， 웅용되고있다.기존인단백질체또는인펩타이들에대한선택적전처리법에는 hydrophilic interaction liquid chromatography(HILIC), strong anion-exchange chromatography (SAX)및 strong cationic ion-exchange chromatography (SCX)등을 사용하여펩타이드들 (Peptides)을분리한후，금속이온의양전하와인산기의 음전하간에화학적결합을이용한 iminobilized metal affinity chromatography (MAC)과 metal oxide affinity chromatography (MOAC)등의전처리를거쳐 인단백질만을추출한후，질량분석기에도입한다.면역학적방법에의한 분석으로는,이차원겔전기영동법 (2 dimensional gel electrophoresis, 2D GE), 면역탁본법 (immunoblot)및면역침강법 (immunoprecipitation, IP)등을사용하여 이루어지고있다.특히，면역탁본법과면역침강법은항체 (Antibody)와 항원 (Antigen)간의결합원리를이용하여겔내에서특정단백질 (targeted protein)을확인할수있는방법이며,상기방법을사용할경우효과적으로 분석하고자하는단백질만을추출할수있다는장점이있다.하지만상기 방법들에서사용되는탁본막 (membrane)또는비즈 (beads)의실라놀기 (silanol group)에항체가모두도포되어있는것이아니므로원하지않은단백질이함께 추출되어추출재현성이낮아지므로,이를방지하기위해서는항체가도포되지 않은실라놀기에알부민을처리하는등다른전처리과정이반드시필요하다. 또한상기추출된단백질을질량분석기에도입시, 2차전처리과정 (추출시 사용된계면활성제의제거또는단백질을펩타이드화하는단계)을거쳐야 하므로복잡한전처리로인한시료손실이발생하는문제점이있다.
발명의상세한설명
기술적과제
[3] 본발명에서는인펩타이드또는인단백질과금속이온간의친화성을이용한 방법의낮은추출효율과복잡한시료전처리과정을개선할수있는마이크로 중공사막효소반웅기기반항원항체반응을이용한온라인항체-특이적 인단백체질량분석법을제공하고자하였다.
과제해결수단
[4] 본발명은 a)세포에존재하는단백질흔합물에환원제를첨가하여변성시킨 후，효소와반응시켜펩타이드흔합물을얻는단계; b) a)단계의펩타이드 흔합물을인단백질또는인펩타이드특이적항체와결합시키는단계;및 c) b)단계에서얻은반웅물을마이크로중공사막효소반웅기 (mHFER)에주입하고 효소처리하여얻은인펩타이드들을추출하여질량스펙트럼을얻는단계;를 포함하는질량분석법에관한것이다.
[5] 상기 a)단계에서사용되는단백질흔합물은제한되지않으나，세포로부터 추출된용출액 (cell lysate)을사용할수있다.본발명에서상기환원제는 제한되지않으나디티오트레이틀 (dithiothreitol, DTT),디티오에리트리톨，트리스 2-카르복시에틸포스핀또는트리부틸포스핀등을사용할수있으며, 바람직하게는디티오트레이를 (DTT)을사용하여단백질의변성을진행할수 있다.
[6] 상기효소는제한되지는않으나단백질분해효소 (protease)라면모두사용할수 있으며，바람직하게는트립신 (trypsin)을사용할수있다. [7] 상기변성후,단백질내시스테인 (cysteine-cysteine)간이황결합 (disulfide bond)의재형성 (refolding)을막기위하여시스테인의티올기 (thiol group, -SH)를 요오드아세트아미드 (iodoacetamide, IAA)를첨가하여알킬화시킴으로써 비가역적형태로변형시킨다.잔류하는 IAA는빛에의한변형또는부가적인 반웅을유도할수있으므로 L-시스테인을첨가하여제거해준다.상기용액을 트립신과단백질간의중량비가 1:40내지 1:60이되도록첨가하여 37 °C에서 18시간동안온도교반기에서반웅시켜효소처리를할수있다.
[8] 상기 b)단계에서인펩타이드또는인단백질특이적항체는제한되지는않지만, 분자량이 50 kDa이상인인세린 (phosphoserine)-,인트레오닌 (phosphothreonine)- 및인티로신 (phosphotyrosine)-항체로이루어진군에서선택되는어느하나의 함체또는둘이상의항체흔합물을사용할수있다.상기인세리-항체는 세린-인산기 (serine-phosphate)와，인트레오닌-항체는트레오닌-인산기 (threonine -phosphate)와，인티로신-항체는티로신-인산기 (tyrosine-phosphate)와각각 친화성을가지고있다.상기인펩타이드또는인단백질과항체의흔합비는 제한되지는않으나，항체또는항체흔합물 100중량부에대하여인단백질또는 인단백질흔합물은 10내지 1000중량부일수있으며，상기반응으로세린-， 트레오닌-및티로신 -인산기와각항체간결합이형성된다.
[9] 상기 c)단계에서사용되는중공사막은제한되지는않으나，바람직하게는
분자량투과한계값이 10 kDa,내경약 200내지 600 μπι,외경약 500내지
ΙΟΟΟμιη인중공사막이좋으며,그재질은폴리스티렌설포네이트，
폴리비닐클로라이드，폴리아크릴나이트릴또는이들의흔합물등으로이루어진 것이좋다.상기추출은제한되지는않으나 4내지 25 °C범위에서수행될수 있으며，본발명의일실시예를통해상기범위내에서추출시에다수의 인펩타이드를획득할수있었다.상기 b)로부터얻어진항체결합펩타이드 흔합물을중공사막효소반응기 (mHFER)에주입하면， 10 kDa이상의크기를 가지는항체결합펩타이드는 mHFER안에머무르게되고，항체와결합하지 않은분자량 10 kDa미만의펩타이드는 mHFER을통해빠져나와용리되게된다. 상기반응은트립신과같은단백질분해효소를주입해줌으로써항체가 분해되면서본기능을잃게되고，이를통하여항체와결합하고있던펩타이드가 용리될수있다.상기펩타이드는역상트랩핑컬럼 (reverse trapping column)에 포집되어소수성정도에따른분리수단인역상 C18컬럼으로분리후， 전자분무이온화 (electrospray ionization, ESI)를거쳐질량분석기로도입되어 분석된다.상기 c)단계의질량분석기는제한되지는않지만,예를들면나노유속 액체크로마토그래피 (nLC, 1260 capillary LC system, Agilent Technologies, Germany)-전기분무이온화-퓨리에변환오비트랩 -텐덤질량분석기 (ESI-FT orbitrap-MS/MS, Q-Exactive, Thermo Scientific, Germany)를사용할수있다.
발명의효과 [10] 본발명에따른온라인인펩타이드또는인단백질추출법은상대적으로낮은 농도로존재하는인산화된템타이드특이적친화력을갖는항체를
사용함으로써효소처리에의해생성된펩타이드흔합물에서항체특이적 인펩타이드들에대한효율적추출과더불어, mHFER장치를이용한자동화된 시스템구성을통해기존복잡한전처리과정에서야기되는문제점들을 최소화하는장점을가지고있다.
도면의간단한설명
[11] 도 1은상기발명된마이크로중공사막효소반웅기 (mHFER)기반
항원항체반응을이용한인단백체추출법에대한모식도이다.
[12] 도 2는세포시료 (MCF7)를이용하여얻어진단백질을효소처리한후，
펩타이드흔합물을항체와반웅시켜중공사막효소반응기기반온 -라인 nLC-ESI-FT orbitrap-MS/MS으로측정한 base peak chromatogram과
질량스펙트럼이다.
[13] 도 3은항체결합인펩타이드또는인단백질추출시， 10 μg항체에대한
단백질의비율별측정된인펩타이드의수이다.
[14] 도 4는도 3과같은경우에대해인펩타이드또는인단백질과항체간의
반웅온도에따라측정된인펩타이드의수이다.
[15] 도 5는확립된조건에따라인세린-항체，인트레오닌 -항체및
인타이로신-항체를인펩타이드와각각반웅시켜측정된인펩타이드의수이다.
[16] 도 6은 MCF7세포용해물로부터얻어진펩타이드흔합물 (10 μ§)중발명된 추출법과기존의인단백체추출법 (FASP, IMAC, Ti02)에의하여추출된 인펩타이드의수이다.
[17] 도 7은도 6으로부터얻어진결과로，추출된인펩타이드들내세린，트레오닌및 티로신에존재하는인산화위치의수이다.
발명의실시를위한최선의형태
[18] 이하본발명을실시예와첨부된도면을참조하여상세히설명한다.그러나 이들은본발명을보다상세하게설명하기위한것으로,본발명의권리범위가 하기의실시예에의해한정되는것은아니다.
[19]
[20] [시험예 1 ]세포용해물 (cell lysate)에존재하는단백질로부터생성된
인펩타이드의추출
[21] MCF7(한국세포주은행，대한민국)세포 (5 * 106 /10 cm dish)를포집하여 0.1 M PBS (phosphate buffered saline)에넣고 tip sonicator를이용하여초음파분쇄한후， 10,000 rpm에서 10분간원심분리하였다.상층액을분리하여，추출된단백질중 100 μδ을분취하여 50 mM중탄산암모늄용액과 10 mM DTT가첨가된용액에 흔합한후， 37 °C에서 2시간동안변성 (denaturation)하였다.상기용액에 270 mM IAA용액을 32 μL첨가하고실온암실에서 30분동안알킬화한후， 400 mM의 L-시스테인을 47 μί첨가하여잔류 ΓΑΑ를제거한다.그후， 2 [xg트립신을넣고 37 °C에서 18시간동안펩타이드화하였다ᅳ상기생성된펩타이드들과
인세린-항체,인트레오닌 -항체및인티로신 -항체흔합물을하기표 1의조성및 온도조건에서흔합반웅시켜항체결합펩타이드혼합물을제조하였다.
[Table 1]
Figure imgf000007_0001
[24] [시험예 2]인펩타이드질량분석법
[25] 온 -라인중공사막효소반웅기 (mHFER)에항체결합펩타이드또는효소의 이동및주입올위해 1내지 10 lJmin이하의유속 (flowrate)으로조절가능한 펌프와온-라인으로시료를주입할수있는시료주입기 (또는 autosampler)가 중공사막의주입구와연결되는구조를가지고있다. mHFER에사용되는 마이크로중공사막 (mHF)은한쪽은에폭시 (epoxy)를사용하여막음으로써 유로의흐름경로가내벽을통해서만이루어지도록하며,상기 mHF의
투과한계는 lO kDa의것으로약 5 의부피를갖도록하였다.
[26] 상기 mHFER에시험예 1에서제조한항체결합펩타이드흔합물을주입한후, 0.1 M PBS를 1내지 5 μϋηώι의유속으로 30분내지한시간동안흘려주었다. 이를통해흔합물내항체와결합하지않은 lO kDa미만크기의펩타이드를 제거한후，중공사막의배출구에역상트랩핑컬럼 (reverse trapping column)을 설치하였다. mHFER내로트립신을주입하고 0.1 M PBS를 1내지 5 [ lJmin의 유속으로 30분내지한시간동안홀려주었다.
[27] mHFER내에포집된 l O kDa이상의항체결합인펩타이드와트립신의
반웅으로항체의분해가유발되어항체와결합하고있던인펩타이드들이 떨어지면서용리된다.상기용리된인펩타이드들은 mHFER의배출구와연결된 역상트랩핑컬럼에포집되고，직접적으로 inoLC-ESI-FT orbitrap-MS MS 기기의유로에연결이되며, binary pump에의한역상용매기을기에따라 C18이 층진된컬럼올통해인펩타이드가갖는소수성정도에따라용리되어 질량분석기로도입되었다.상기일련의과정을도 I에도식화하였고,질량분석 결과의예를도 2에도식화하였다.
[28] 실시예 1내지실시예 3은단백질또는펩타이드와항체간의중량비에따른 추출된인단백질및인펩타이드수에관한내용으로，도 3의결과에따라 펩타이드와항체간의중량비가 1:10, 1:1및 10:1일때,측정된인펩타이드의 수는각각 400개, 553개및 229개로나타났으며，이를통하여중량비를 1: 1로 사용하였을때추출된인펩타이드수가가장높게나타나는것을확인하였다.
[29] 실시예 2및실시예 4의조건으로추출효율을측정한결과,도 4와같이
반웅온도가 25 °C일경우에 553개의인펩타이드가확인되었고， 4 °C일경우에는 296개의인펩타이드가확인되었다.따라서반웅온도를 25 °C로하였을때추출된 인펩타이드의수가증가한것을확인하였다.
[30] 실시예 5내지실시예 7은단백질또는펩타이드와각각의항체 (인세린 -항체， 인트레오닌 -항체및인티로신-항체에대하여출처가다른항체를 2개씩사용)를 반응시켜비교한결과로,도 5에서나타난것과같이추출하기전에측정한 인펩타이드의수는 272개이며，인세린 -항체 I과 Π을사용하여측정한
인펩타이드의수는 1,438개와 1 ,229개로측정되었다.또한인트레오닌 -항체 I과 II에서는 1,397개와 202개로측정되었으며,인티로신 -항체 I과 II에의하여 추출된인펩타이드의수는 713개와 76개인것으로확인되었다.이결과를 토대로추출하지않은것에비하여각항체를사용하였을때측정된
인펩타이드의수가증가한것을확인하였고，특히인세린 -펩타이드가가장많이 측정되었으며,인트레오닌 -펩타이드，인티로신-펩타이드순으로많이측정되는 것을확인하였다.
[31]
[32] [시험예 3]인펩타이드추출법에따른효율측정
[33] MCF7세포용해물로부터얻어진단백질을효소처리한후， 10 g의펩타이드 흔합물을사용하여기존에보고된인펩타이드의추출방법 [filter aided sample preparation (FASP), immobilized metal affinity chromatography (IMAC), titanium dioxide (Ti02)]과본발명을통하여인단백질의추출효율을비교하였다.
[34] FASP추출방범
[35] FASP는 10 의펩타이드흔합물과항체 10 을반응시켜투과한계 10 kDa의 centrifugal filter (Millipore, Ireland)로옮긴후， 200 의 0.1 M PBS를첨가하여 흔합한다음 14,000 x g에서 10분간원심분리하였다.이과정을두번반복하여 항체와결합하지않은펩타이드들을제거하고, 0.2 의트립신 (항체：효소 =50:1， w/w)을넣어 37도에서 18시간동안반웅시켰다.효소처리된흔합물에 200 의 0.1 M PBS를첨가하여흔합한후, 14,000 x g에서 10분간원심분리하여 항체로부터분리된인펩타이드를수집하였다.상기과정을 1회추가로반복하여 인펩타이드를수집하고，수집된용액은진공농축기를이용하여농축시킨후 질량분석기에도입하여측정하였다.
[36] TMAC추출방법
[37] IMAC을이용한인펩타이드의추출을위하여， Ni-NTA spin column (Qiagen, Hilden, Germany)를사용하였다.상기 Ni-NTA beads는한쪽끝이 3 mm의 다공성유리재질의 sol-gel frits으로막힌 capiUary (내경 200 um,길이 100 mm)에 층진하여사용하였다.시린지펌프,시료주입기및 Ni-NTA beads가층진된 capillary순으로연결하고, 1내지 5 μ!7πώι의유속으로용매를홀려주어시료가 도입될수있도록장치하였다. O.l M NaCl용액에녹인 50 mM EDTA용액 100 [xL를흘려주어 Ni-NTA beads에존재하는 Ni2+이온을제거하고， 100 의 0.2 Μ FeCl3용액을흘려주어 NTA beads에 Fe3+를첨가하여활성화하였다.시료주입 전,상기 capillary에 loading buffer (6 %아세트산을함유한 0.1 M NaOH용액， pH 3.6)을 30분간흘려주어 NTA beads를평형화하였다.시료주입기를통하여 10 의펩타이드흔합물을주입한후, loading buffer로 20분간홀려줌으로써 Fe-NTA beads에인단백질또는인펩타이드들이킬레이트화할수있도록하였다. 킬레이트화하지않은펩타이드들을제거하기위해서상기 capillary에 wasing buffer (loading buffer/acetontrile, 75/25, v/v)를흘려주어상기 capillary에서 킬레이트화하지않은펩타이드들을제거하였다.킬레이트화된인단백질을 용출하기전， 100 의로딩버퍼 (loading buffer)를홀려주어평형화시켜주고， pH 11의 4 % NH4OH용액을홀려주어킬레이트화된펩타이드들을용출시켜 수집하였다.상기수집된용액은진공농축기를이용하여건조한후， 0.1% 포름산 (formic acid)용액에재용해시킨후질량분석기에도입하여측정하였다.
[38] TiQ2추출방범
[39] Ti02를이용한인펩타이드추출을위하여, Ti02 beads가층진된
고체상추출 (solid phase extration, SPE)카트리지 (GL Science, Japan, 50mg/3mL)를 이용하였다.상기카트리지는원심분리기를이용할수있도록 15 mL튜브속에 위치시켰다.건조된 SPE카트리지를활성화하기위하여 0.5% triflouroacetic acid를함유한 buffer A (acetonitrile/water=80/20, v/v) 200 를넣고， 200 x g에서 2분간원심분리한후， buffer B (lactic acid/buffer A=300mg/mL, w/v) 200 를넣고 200 X g에서 2분간원심분리하여평형화하고，류브에수집된용액은버렸다. 10 Hg의펩타이드혼합물을 Ti02카트리지에넣고시료 500 당 1000 의 buffer B를카트리지안에서흔합한후, 200 x g에서 5분간원심분리하였다.류브에 수집된용액을다시 Ti02카트리지안에넣고동일조건으로원심분리하며，상기 과정을 10회반복하여 Ti02 beads에더많은인펩타이드들이킬레이트화되도록 하였다 . Ti02카트리지에인펩타이드외다른펩타이드들을제거하기위하여， 200 nL의 buffer B를이용하여 200 x g에서 2분간원심분리하고, 200 의 buffer A로 200 x g에서 2분간원심분리하여세척하였다.인펩타이드들을용출시키기 위하여 Ti02카트리지를새로운 15 mL류브로옮기고， 5 %암모늄 (ammonium) 수용액 200 μΐ^를사용하여 200 x g에서 5분간원심분리하고， 200 의 5 % pyrrolidine수용액을사용하여동일조건에서원심분리하여인펩타이드를용리한 후,수집된두용액을흔합하여진공농축기로건조시켰다.상기건조된시료는 0.1% formic acid에재용해시켜질량분석기에도입하여측정하였다.
[40] 기존 3가지추출방법과 mHFER름이욘하방 의비교 과
[41] 도 6은상기언급된 3가지의추출방법과본발명을통하여추출된
인펩타이드의수를나타낸것으로,동일한 10 μ 의펩타이드들을사용하여 실험을진행하였을때，항체흔합물 (phospho-specific antibodies, pAbs)을이용한 방법은 773개， FASP는 149개， IMAC은 18개, Ti02는 2개의인펩타이드를각각 추출한것으로나타났으며,기존추출방법인 FASP를사용할때보다
항체흔합물을사용할때놀랍게도 5배이상의인펩타이드들을추출하는것을 확인할수있었다.
[42] 도 7은도 6의실험을통하여인펩타이드내세린，트레오닌및티로신위치에 존재하는인산화의수를나타낸것이다.항체흔합물을이용한방법은
인펩타이드내 1,585개， FASP는 189개, IMAC은 24개, Ti02는 2개의인산화 위치를나타내었다.상기결과로부터기존방법인 FASP에비해항체흔합물을 이용한추출방법이놀랍게도 8배이상더많은인산화위치를나타내는것을 확인할수있었다.
[43] 또한,도 6의인펩타이드수와도 7의인산화수를비교해보면본발명의
항체흔합물추출방법을사용하였을때단일인펩타이드들뿐만아니라종래에 확인할수없었던이중및삼중인펩타이드를포함하는다증인펩타이드까지 추출할수있음을확인하여,본발명을완성하였다.

Claims

청구범위
a)세포에존재하는단백질흔합물에환원제를첨가하여변성시킨 후，효소와반웅시켜펩타이드흔합물을얻는단계;
b) a)단계의펩타이드흔합물을인단백질또는인펩타이드특이적 항체와결합시키는단계;및
c) b)단계에서얻은반응물을마이크로중공사막효소
반웅기 (mHFER)에주입하고효소처리하여얻은인펩타이드들을 추출하여질량스펙트럼을얻는단계；를포함하는질량분석법. 제 1항에있어서,
상기환원제는디티오트레이톨 (dithiothreitol, DTT),
디티오에리트리를,트리스 2-카르복시에틸포스핀또는트리부틸 포스핀인것을특징으로하는질량분석법.
제 1항에있어서，
상기효소는단백질분해효소 (protease)인것을특징으로하는 질량분석법.
제 3항에있어서,
상기단백질분해효소는트립신 (trypsin)인것을특징으로하는 질량분석법.
제 1항에있어서,
상기인단백질또는인펩타이드특이적항체는
인세린 (phosphoserine)-항체，인트레오닌 (phosphothreonine)-항체및 인티로신 (phosphotyrosine)-항체로이루어진군에서선택되는어느 하나의항체또는둘이상의항체혼합물인것을특징으로하는 질량분석법.
제 1항에있어서，
상기항체또는항체흔합물 100중량부에대하여인단백질또는 인단백질흔합물은 10내지 1000중량부인것을특징으로하는 질량분석법.
제 1항에있어서，
상기추출은 4내지 25 °C범위에서수행되는것을특징으로하는 질량분석법.
PCT/KR2015/008641 2014-08-19 2015-08-19 마이크로 중공사막 효소 반응기 - 텐덤 질량분석법을 이용한 단세포군 항체 기반 온라인 인단백질 프로테오믹스 분석방법 WO2016028075A1 (ko)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US15/504,732 US20170261513A1 (en) 2014-08-19 2015-08-19 Monoclonal antibody based online phosphoprotein proteomics analysis method using microbore hollow fiber enzymatic reactor-tandem mass spectrometry

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020140107564A KR101675303B1 (ko) 2014-08-19 2014-08-19 마이크로 중공사막 효소 반응기-텐덤 질량분석법을 이용한 단세포군 항체 기반 온라인 인단백질 프로테오믹스 분석방법
KR10-2014-0107564 2014-08-19

Publications (1)

Publication Number Publication Date
WO2016028075A1 true WO2016028075A1 (ko) 2016-02-25

Family

ID=55350955

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2015/008641 WO2016028075A1 (ko) 2014-08-19 2015-08-19 마이크로 중공사막 효소 반응기 - 텐덤 질량분석법을 이용한 단세포군 항체 기반 온라인 인단백질 프로테오믹스 분석방법

Country Status (3)

Country Link
US (1) US20170261513A1 (ko)
KR (1) KR101675303B1 (ko)
WO (1) WO2016028075A1 (ko)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106198319A (zh) * 2016-06-23 2016-12-07 中国科学院南京地理与湖泊研究所 一种基于dgt同步测定8种氧化型阴离子的方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11726096B2 (en) * 2018-10-04 2023-08-15 Regeneron Pharmaceuticals, Inc. Fast protein sequencing

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004503780A (ja) * 2000-06-12 2004-02-05 ユニバーシティ　オブ　ワシントン リンペプチドの選択的標識および単離ならびにプロテオーム分析への適用
JP2006515674A (ja) * 2004-02-10 2006-06-01 コリア ベーシック サイエンス インスティテュート リン酸化タンパク質の質量分析及びリン酸化位置分析用標識物質
KR20090087750A (ko) * 2008-02-13 2009-08-18 이화여자대학교 산학협력단 인산화 타이로신 잔기에 대한 항체
KR20130030464A (ko) * 2011-09-19 2013-03-27 한국표준과학연구원 중공사막을 이용한 단백질의 효소 처리장치 및 이를 이용한 온-라인 프로테오믹스 방법
KR20140042407A (ko) * 2012-09-28 2014-04-07 한국표준과학연구원 안정동위원소 표식 펩타이드의 정량 분석용 키트 및 이를 이용한 정량방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004503780A (ja) * 2000-06-12 2004-02-05 ユニバーシティ　オブ　ワシントン リンペプチドの選択的標識および単離ならびにプロテオーム分析への適用
JP2006515674A (ja) * 2004-02-10 2006-06-01 コリア ベーシック サイエンス インスティテュート リン酸化タンパク質の質量分析及びリン酸化位置分析用標識物質
KR20090087750A (ko) * 2008-02-13 2009-08-18 이화여자대학교 산학협력단 인산화 타이로신 잔기에 대한 항체
KR20130030464A (ko) * 2011-09-19 2013-03-27 한국표준과학연구원 중공사막을 이용한 단백질의 효소 처리장치 및 이를 이용한 온-라인 프로테오믹스 방법
KR20140042407A (ko) * 2012-09-28 2014-04-07 한국표준과학연구원 안정동위원소 표식 펩타이드의 정량 분석용 키트 및 이를 이용한 정량방법

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106198319A (zh) * 2016-06-23 2016-12-07 中国科学院南京地理与湖泊研究所 一种基于dgt同步测定8种氧化型阴离子的方法

Also Published As

Publication number Publication date
KR101675303B1 (ko) 2016-11-11
KR20160022036A (ko) 2016-02-29
US20170261513A1 (en) 2017-09-14

Similar Documents

Publication Publication Date Title
Motoyama et al. Multidimensional LC separations in shotgun proteomics
Ahmed Sample preparation and fractionation for proteome analysis and cancer biomarker discovery by mass spectrometry
Kašička Recent developments in capillary and microchip electroseparations of peptides (2011–2013)
Ma et al. Immobilized enzyme reactors in proteomics
US6814870B2 (en) Microchip electrospray device and column with affinity adsorbents and use of the same
Dong et al. Coupling strong anion-exchange monolithic capillary with MALDI-TOF MS for sensitive detection of phosphopeptides in protein digest
Pernemalm et al. Affinity prefractionation for MS‐based plasma proteomics
Xiu et al. Effective protein separation by coupling hydrophobic interaction and reverse phase chromatography for top-down proteomics
Kašička Recent developments in capillary and microchip electroseparations of peptides (2017–mid 2019)
JP2005529335A (ja) 開放チャンネルを使って生体分子を固体相として抽出するシステムと方法
CN112996580B (zh) 串联配对柱化学性质用于高通量蛋白质组学外泌体分析
JP7412407B2 (ja) グリコシル化ペプチドの検出及び定量
Olszowy et al. Urine sample preparation for proteomic analysis
Hedrick et al. Digestion, purification, and enrichment of protein samples for mass spectrometry
US20160377635A1 (en) Measurement of Oxytocin and Vasopressin
Molnarova et al. Liquid chromatography and capillary electrophoresis in glycomic and glycoproteomic analysis
Maráková et al. Capillary electrophoresis‐mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018–March 2023)
Seyfinejad et al. Capillary electrophoresis-mass spectrometry in pharmaceutical and biomedical analyses
Kuzyk et al. CE-MS for proteomics and intact protein analysis
Ribeiro et al. Recent stationary phase‐based fractionation strategies in proteomic analysis
Núñez et al. Novel functionalized nanomaterials for the effective enrichment of proteins and peptides with post-translational modifications
WO2016028075A1 (ko) 마이크로 중공사막 효소 반응기 - 텐덤 질량분석법을 이용한 단세포군 항체 기반 온라인 인단백질 프로테오믹스 분석방법
Bakry et al. Derivatized nanoparticle coated capillaries for purification and micro-extraction of proteins and peptides
Choi Reaching for the deep proteome: Recent nano liquid chromatography coupled with tandem mass spectrometry-based studies on the deep proteome
Štěpánová et al. CE–MS Approaches for Peptidomics

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15833606

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 15504732

Country of ref document: US

122 Ep: pct application non-entry in european phase

Ref document number: 15833606

Country of ref document: EP

Kind code of ref document: A1