WO2020215791A1 - Sucre ou groupe de sucre bionique marqué par un isotope, procédé de préparation correspondant et utilisation associée - Google Patents

Sucre ou groupe de sucre bionique marqué par un isotope, procédé de préparation correspondant et utilisation associée Download PDF

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WO2020215791A1
WO2020215791A1 PCT/CN2019/130253 CN2019130253W WO2020215791A1 WO 2020215791 A1 WO2020215791 A1 WO 2020215791A1 CN 2019130253 W CN2019130253 W CN 2019130253W WO 2020215791 A1 WO2020215791 A1 WO 2020215791A1
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sugar
sugar chain
isotope
sample
group
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PCT/CN2019/130253
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Chinese (zh)
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顾建新
任士芳
秦文俊
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复旦大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/005—Sugars; Derivatives thereof; Nucleosides; Nucleotides; Nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • 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
    • G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled

Definitions

  • This application belongs to the fields of biotechnology, analytical chemistry technology and medicine. Specifically, this article relates to isotope-labeled biomimetic sugars or sugar groups, their preparation methods and applications, and in particular to a simple, high-throughput and accurate stable isotope internal standard sugar group analysis method.
  • Glycosylation is a ubiquitous post-translational modification that not only affects the structure, solubility and stability of proteins, but also involves multiple biological processes, such as protein folding, cell recognition, and binding of receptors to ligands. Studies have reported that about 50% of mammalian proteins are glycosylated. Abnormal glycosylation of glycoproteins is inseparable from many diseases, including arthritis, congenital diseases, and tumor development and metastasis.
  • glycomic-based biomarkers in body fluids such as serum, plasma, and urine.
  • Many glycoproteins have been widely used in clinical diagnosis and treatment of diseases, such as cancer antigen 125 (CA125), carcinoembryonic antigen (CEA), prostate specific antigen (PSA) and so on. Therefore, analysis and research on glycosylation of disease-related glycoproteins will help to fully understand the occurrence and development of various physiology and pathology, and realize its practical application value in disease diagnosis and treatment.
  • CA125 cancer antigen 125
  • CEA carcinoembryonic antigen
  • PSA prostate specific antigen
  • sugar chains can be used not only for disease detection, but also for many other aspects, such as antibody drug research.
  • Different glycosylation modified antibody drugs have different biological functions and have different effects in disease treatment.
  • Quantitative methods based on biological mass spectrometry can generally be divided into two categories: non-isotopic labeling quantitative methods and isotope labeling quantitative methods.
  • the non-isotopic labeling quantitative method is a simple quantitative method that separates sugar chains from different samples, carries out a series of derivatization treatments, and then carries out mass spectrometry detection and analysis. By comparing the strength or weakness of the mass spectrum peak signal Peak area to obtain quantitative results of sugar chain expression in different samples.
  • non-isotope-labeled quantitative glycomics method has the advantages of simple operation, no change in sample structure, and low experimental cost, the matrix effect, mass spectrometry response, and operating errors can lead to low accuracy, low reproducibility, etc. of sugar chain analysis.
  • the quantitative results have large errors.
  • the isotope labeling quantitative method is a more accurate quantitative method.
  • the sugar chains in different samples are labeled, and then mixed for mass spectrometry detection and analysis.
  • this method one mass spectrum can display all samples, and the quantitative results can be obtained by comparing the intensity or peak area of the paired mass spectrum peak signals.
  • the commonly used quantitative methods of isotope labeling include: enzymatic hydrolysis to introduce isotope labeling, metabolic introduction of isotope labeling, and chemical derivatization to introduce isotope labeling.
  • This article provides an isotope-labeled bionic sugar or sugar group, its preparation method and application. Another focus of this article also provides the application of the lung cancer sugar chain markers identified by the method herein and the substance for detecting the lung cancer sugar chain markers in the preparation of products for lung cancer diagnosis and/or lung cancer treatment plan screening .
  • a modified isotope-labeled biomimetic sugar or a sugar group comprising a modified isotope-labeled biomimetic sugar, wherein, compared with its corresponding unmodified glycan, the biomimetic sugar includes a reduced sugar chain The terminal alcoholic hydroxyl group and isotope labeling, and the molecular weight of the biomimetic sugar is increased by 3 Daltons or more.
  • the biomimetic sugar has the same sugar chain composition and abundance as its corresponding unmodified sugar.
  • the reducing end of the unmodified glycan is a hemiacetal group.
  • the alcoholic hydroxyl group and the isotope-labeled reducing end of the unmodified glycan are produced by ring opening through a reduction reaction.
  • the reducing ends of the unmodified glycan and the biomimetic sugar are as shown in formula (I) and formula (I′), respectively, wherein Represents the bond to other parts of the sugar chain, D represents deuteration:
  • the biomimetic sugar compared with the corresponding natural polysaccharide, includes an alcohol hydroxyl group at the reducing end of the sugar chain and an isotope label, and the molecular weight of the biomimetic sugar is increased by 3 Daltons or more.
  • the reduction reaction is performed using sodium borodeuteride (NaBD 4 ).
  • the sugar chain is an N-sugar chain or an O-sugar chain.
  • the sugar chain to be modified includes one or more sugar chains.
  • the sugar chains to be modified are obtained from natural samples.
  • the sugar chain to be modified is obtained from a sample to be tested.
  • the sample is selected from: body fluid samples, such as blood, serum, plasma, urine, saliva, lymph, spinal fluid, ascites, and amniotic fluid; cell samples, such as cell samples isolated from tissues, in vitro culture Cell samples; tissue samples, such as cancer tissue, para-cancerous tissue, normal tissue, in the form of fresh tissue samples, immobilized tissue samples; production or development samples, such as quality inspection samples with sugar chain drugs (such as antibody drugs) , Antibody drug development samples.
  • body fluid samples such as blood, serum, plasma, urine, saliva, lymph, spinal fluid, ascites, and amniotic fluid
  • cell samples such as cell samples isolated from tissues, in vitro culture Cell samples
  • tissue samples such as cancer tissue, para-cancerous tissue, normal tissue, in the form of fresh tissue samples, immobilized tissue samples
  • production or development samples such as quality inspection samples with sugar chain drugs (such as antibody drugs) , Antibody drug development samples.
  • the sugar chain to be modified is a sugar chain released from a sugar complex.
  • the sugar chain is released using PNGase F, Endoglycosidase H, F2, F3, Endoglycosidase II), chemical methods (such as ⁇ elimination reaction), and/or combinations thereof. .
  • the method further includes protecting the sialic acid on the sugar chain, such as esterification protection.
  • bionic sugar chain corresponding to a sample sugar chain, the bionic sugar chain including an alcohol hydroxyl group at the reducing end of the sugar chain and an isotope label, and compared with the sample sugar chain, the molecular weight of the bionic sugar is increased by 3 daltons Meal or more;
  • the sample is selected from: body fluid samples, such as blood, serum, plasma, urine, saliva, lymph, spinal fluid, ascites, and amniotic fluid; cell samples, such as cell samples isolated from tissues, in vitro culture Cell samples; tissue samples, such as cancer tissue, para-cancerous tissue, normal tissue, in the form of fresh tissue samples, immobilized tissue samples; production or development samples, such as quality inspection samples with sugar chain drugs (such as antibody drugs) , Antibody drug development samples.
  • body fluid samples such as blood, serum, plasma, urine, saliva, lymph, spinal fluid, ascites, and amniotic fluid
  • cell samples such as cell samples isolated from tissues, in vitro culture Cell samples
  • tissue samples such as cancer tissue, para-cancerous tissue, normal tissue, in the form of fresh tissue samples, immobilized tissue samples
  • production or development samples such as quality inspection samples with sugar chain drugs (such as antibody drugs) , Antibody drug development samples.
  • the biomimetic sugar is prepared using the method described herein.
  • the method includes:
  • step (i) and/or step (ii) a sample sugar chain whose reducing end is hemiacetal is provided by releasing the sugar chain from the sugar complex or a biomimetic sugar chain is obtained through reduction labeling;
  • step (ii) through a reduction reaction, the reducing end hemiacetal structure of the sample sugar chain or sugar group is converted into an alcoholic hydroxyl group and contains an isotope label; preferably by a reduction reaction using sodium borodeuteride (NaBD 4 ), The reducing end hemiacetal structure of the sample sugar chain is converted into an alcohol hydroxyl group and deuterated; and/or
  • the mass analysis of step (iv) is performed by one or more methods selected from the following group: mass spectrometry (MS) analysis, such as matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS, such as matrix-assisted laser desorption ionization-time of flight Mass spectrometry (MALDI-TOF-MS), matrix-assisted laser desorption ionization-quaternary ion trap-time-of-flight mass spectrometry (MALDI-QIT-TOF MS)), electrospray mass spectrometry (ESI-MS), fast atom bombardment mass spectrometry (FAB- MS), cascade mass spectrometry, multistage mass spectrometry, electrospray-collision induced dissociation mass spectrometry (ESI-CID-MS); high performance liquid chromatography HPLC; liquid chromatography mass spectrometry (LC-MS); capillary electrophoresis-mass spectrometry ( CE-MS); and
  • the comparison and/or ratio in step (v) includes: peak position comparison, peak height comparison, peak area comparison and/or ratio, and any combination thereof, such as comparison of peak areas of paired peak signals, sample sugar chains The ratio of peak area/internal standard sugar chain peak area (light/heavy); and/or
  • the internal standard sugar chain and the sample sugar chain are processed (preferably the same treatment) to adapt to subsequent quality analysis, such as purification, enrichment, dilution of sugar chains, or the esterification of sugar chains to protect sugars Sialic acid at the end of the chain.
  • the sugar complex is selected from glycoproteins, proteoglycans, glycopeptides, glycolipids, or any combination thereof, such as sugar chain-containing antibodies.
  • enzymatic methods such as PNGase F, Endoglycosidase H, F2, F3, endoglycosidase II
  • chemical methods such as ⁇ elimination reaction
  • the purification and/or enrichment is performed by centrifugation, precipitation separation, filtration, chromatographic separation and the like.
  • the comparison and/or ratio are obtained through calculation software and/or algorithms.
  • each sugar chain that is not isotopically labeled in the sample has a corresponding isotope-labeled sugar chain.
  • the method includes:
  • terminal sialic acid protection is performed on the isotope-labeled sugar chain and the sugar chain that is not isotope-labeled, and the obtained sialic acid protected sugar chain may be optionally purified and/or enriched;
  • the method is further used to:
  • Sugar group quantitative and/or qualitative analysis for example, for disease diagnosis and/or prognosis judgment based on sugar chain markers (such as cancer antigen 125 (CA125), carcinoembryonic antigen (CEA), prostate specific antigen (PSA)); Potential disease-related sugar chain markers; development and/or quality control of sugar complexes (such as sugar chain drugs, such as antibodies containing glycosylation modification); protein glycosylation modification analysis.
  • sugar chain markers such as cancer antigen 125 (CA125), carcinoembryonic antigen (CEA), prostate specific antigen (PSA)
  • Potential disease-related sugar chain markers such as sugar chain drugs, such as antibodies containing glycosylation modification
  • protein glycosylation modification analysis such as sugar chain drugs, such as antibodies containing glycosylation modification
  • the sugar chains or sugar groups herein and/or the reagents and/or devices used in the methods described herein are being prepared for use in sugar chain marker-based disease diagnosis and/or prognostic judgment and screening potential Disease-related sugar chain markers, sugar complexes (such as sugar chain drugs, such as glycosylation-modified antibodies) development and/or quality control, application of protein glycosylation modification analysis products.
  • sugar chain marker-based disease diagnosis and/or prognostic judgment and screening potential Disease-related sugar chain markers sugar complexes (such as sugar chain drugs, such as glycosylation-modified antibodies) development and/or quality control, application of protein glycosylation modification analysis products.
  • lung cancer sugar chain markers selected from the following group are provided:
  • H represents hexose
  • N represents N-acetylglucosamine
  • F represents fucose
  • E represents ⁇ 2,6-linked sialic acid.
  • this article there is provided the application of a substance for detecting the above-mentioned lung cancer sugar chain marker in the preparation of a product for diagnosis of lung cancer and/or screening of a lung cancer treatment plan.
  • a method for diagnosing lung cancer and/or screening for a treatment plan for lung cancer comprising detecting the level of a lung cancer sugar chain marker as described above in a sample.
  • kits for lung cancer diagnosis and/or lung cancer treatment plan screening which includes detecting one or more of the aforementioned sugar chain markers in a sample.
  • the detection is carried out using the isotope-labeled biomimetic sugar or a sugar panel containing isotope-labeled biomimetic sugar as described herein, an analysis method or a product.
  • FIG 1 Schematic diagram of the flow of an embodiment of this application.
  • Figure 2 (A) The upper figure shows the mass spectrum of NA2G1F sugar chains without reduction labeling, and the lower figure shows the mass spectrum of bionic NA2G1F sugar chains after reduction labeling;
  • Figure 2(B) Mass spectrum of NA2G1F sugar chain mixture before and after reduction labeling.
  • Part B of Figure 3 Mass spectrum of the bionic sugar chain after reduction labeling.
  • Figure 4 Linear analysis of the peak area ratio of N-glycan standard NA2G1F and internal standard.
  • Figure 5 Linear analysis of the peak area ratio of H3N4F1 and H5N4F1E1 glycotypes and internal standard on glycoprotein standard IgG.
  • Figure 6 Mass spectrum of an exemplary human serum N-sugar group and its bionic sugar group mixture.
  • This application provides a new method for glycan analysis based on stable isotope labeling (such as 1 H/ 2 D labeling) internal standard.
  • the application also provides a bionic sugar group based on stable isotope labeling (such as 1 H/ 2 D labeling) internal standard.
  • stable isotope labeling such as 1 H/ 2 D labeling
  • the internal standard that is, the bionic sugar (group) and the unmodified sugar (group) have the same sugar chain composition and similar sugar chain abundance distribution. Therefore, this method not only retains the advantages of simple operation of the non-isotope-labeled quantitative glycomics method, but also has the advantages of accurate isotope-labeled glycomic analysis methods.
  • the inventors respectively used sugar chains and glycoprotein standards to investigate the linear relationship and coefficient of variation of the method, and the results showed that the stable isotope internal standard sugar group analysis method of the present application has a good linear relationship within two orders of magnitude dynamic range, and The coefficient of variation is smaller than the prior art method.
  • the inventor further used the method of this application to analyze the sugar group in the serum sample, and investigated the same-day and day-to-day reproducibility of the method. The results show that the method of the present invention has excellent same-day reproducibility and day-to-day reproducibility. The availability and the coefficient of variation are significantly lower than the prior art methods.
  • the method and the biomimetic sugar set described in this article have the advantages of simple operation, time saving, and lower experimental cost.
  • the inventors have identified lung cancer-specific glycan changes through quantitative analysis of sugar chains in serum samples, thereby further demonstrating the feasibility of this quantitative method.
  • containing, “having” or “including” includes “including”, “consisting essentially of”, “consisting essentially of”, and “consisting of. 7-8 constitute”; “mainly constituted by”, “basically constituted by " and “made by " belong to “contains” and “has “Or “include” subordinate concept.
  • the method herein can be used to analyze various samples containing sugar chains, the samples include but not limited to: body fluid samples, such as blood, serum, plasma, urine, saliva, lymph, spinal fluid, ascites, amniotic fluid; cell samples, Such as cell samples isolated from tissues, cell samples cultured in vitro; tissue samples, such as cancer tissue, para-cancerous tissue, and normal tissue, in the form of fresh tissue samples, immobilized tissue samples; production or development samples, such as sugar chains Quality control samples of drugs (such as antibody drugs), antibody drug development samples; etc.
  • body fluid samples such as blood, serum, plasma, urine, saliva, lymph, spinal fluid, ascites, amniotic fluid
  • cell samples Such as cell samples isolated from tissues, cell samples cultured in vitro
  • tissue samples such as cancer tissue, para-cancerous tissue, and normal tissue, in the form of fresh tissue samples, immobilized tissue samples
  • production or development samples such as sugar chains Quality control samples of drugs (such as antibody drugs), antibody drug development samples; etc.
  • glycoprotein refers to all sugar chains expressed in a sample (such as cells, tissues) or all sugar chains on a specific type of glycoprotein.
  • sample sugar chain As used herein, the terms “sample sugar chain”, “sample sugar chain to be tested”, “unmodified sugar chain” and “sugar chain not labeled with isotope” are used interchangeably, and all refer to the need to analyze the sugar chain.
  • the sugar chains present in the sample can be processed by sugar chain release (such as enzymatic hydrolysis, chemical release), purification, enrichment, derivatization and other steps for quality analysis, but it does not need to be labeled with isotope.
  • internal standard sugar (chain) As used herein, “internal standard sugar (chain)”, “biomimetic sugar (chain)”, “modified sugar chain”, “isotope-labeled reducing sugar (chain)” and “isotope-labeled bionic sugar (chain)” are interchangeable Use refers to the sugar chain standard product that has been reduced by isotope labeling as described herein, or relative to the “sample sugar chain", it is from the same sample or the same species source and subjected to the same treatment, except that the sugar chain has undergone the isotope reduction labeling step. Chain substance.
  • the internal standard sugar chain is prepared from the sample to be tested and is a sugar chain mixture, which can be used as an internal standard sugar chain library.
  • each sugar chain in the sample has a corresponding internal standard, which makes the quantification more accurate and is more conducive to the analysis of large samples.
  • the biomimetic sugar (group) and the unmodified sugar (group) have the same sugar chain structure and similar sugar chain abundance distribution, which is conducive to accurate analysis of the sugar chains of the sample.
  • the internal standard sugar chain and the sugar chain to be tested can be any N-sugar chain or O-sugar chain of interest, including but not limited to: sugar chains as disease markers, such as cancer antigens (such as CA125, CA242, CA 19- 9. CA15-3, etc.), carcinoembryonic antigen (CEA), prostate specific antigen (PSA), etc.; sugar chains carried by sugar chain drugs, such as sugar chains carried by antibody drugs (such as trastuzumab); Important sugar chains that affect biological processes, such as sugar chains that affect signal transmission, cell growth and development, immune cell regulation, tumor occurrence and development.
  • cancer antigens such as CA125, CA242, CA 19- 9. CA15-3, etc.
  • CEA carcinoembryonic antigen
  • PSA prostate specific antigen
  • sugar chains carried by sugar chain drugs such as sugar chains carried by antibody drugs (such as trastuzumab)
  • Important sugar chains that affect biological processes such as sugar chains that affect signal transmission, cell growth and development, immune cell regulation, tumor occurrence and development.
  • the sugar chain described herein may be N-sugar chain or O-sugar chain, preferably N-sugar chain.
  • the sugar chains described herein may be free sugar chains or sugar chains released from sugar complexes.
  • the term "sugar chain reducing end" refers to the end of the glycan having a free hemiacetal hydroxyl group.
  • the reducing end of the glycan may be a hemiacetal.
  • the sugar chain of terminal hemiacetal can be obtained by techniques known in the art.
  • an enzymatic method can be used to release sugar chains.
  • the available enzymes include but are not limited to: PNGase F, Endoglycosidase H, F2, F3, Endoglycosidase II or any combination thereof; chemical Methods to release sugar chains, such as ⁇ elimination reaction; also can use a combination of enzymatic and chemical methods to release sugar chains.
  • the sugar chain to the N-, the compounds may be employed deuterium labeled internal standard sugar chain ends, such as hydroxy 2 D in order to obtain labeled, so that the molecular weight of the resulting sugar chain standard isotopically labeled samples than unlabeled sugar The chain increases by 3 Da.
  • NaBD 4 can be used as a reducing reagent and H 2 18 O as a solvent when ⁇ is eliminated, so that the molecular weight of the obtained isotope-labeled internal standard sugar chain is higher than that of the unlabeled sample.
  • the sugar chain increases by 3Da.
  • the sugar chain may optionally be derivatized, for example, to improve the sensitivity of mass spectrometry detection or to protect the terminal group of the sugar chain.
  • Derivatization can include, but is not limited to: methylamination, esterification, methylation, acetylation, reductive amination, and the like.
  • the type and timing of derivatization can be selected according to needs. For example, usually esterification derivatization is performed after isotope labeling.
  • sugar chains After the sugar chains are subjected to any treatment, they can be purified and/or enriched using techniques known in the art.
  • the sugar chains can be purified and/or enriched after the sugar chains are released, after isotopic labeling of the internal standard sugar chains, and/or after the sugar chains are derivatized.
  • Methods of purification and/or enrichment may include, but are not limited to: centrifugation, filtration, adsorption, chromatography and the like.
  • the two After obtaining the isotope-labeled internal standard sugar chain and the same treatment but not the isotope-labeled sample sugar chain, the two can be mixed in the required ratio for quality analysis.
  • mass analysis of the mixture can be carried out in a suitable manner.
  • MS mass spectrometry
  • MALDI MS matrix-assisted laser desorption ionization mass spectrometry
  • MALDI-TOF-MS matrix-assisted laser desorption ionization-time of flight Mass spectrometry
  • MALDI-QIT-TOF MS matrix-assisted laser desorption ionization-quaternary ion trap-time-of-flight mass spectrometry
  • electrospray mass spectrometry ESI-MS
  • FAB- MS fast atom bombardment mass spectrometry
  • cascade mass spectrometry multistage mass spectrometry
  • electrospray-collision induced dissociation mass spectrometry EI-CID-MS
  • high performance liquid chromatography HPLC liquid chromatography mass spectrometry
  • LC-MS liquid chromatography mass spectrometry
  • capillary electrophoresis-mass spectrometry capillary electrophores
  • the quality analysis data can be further calculated and processed to obtain the required sugar group related information. For example, you can compare the peak position, peak height, peak area and any combination of the sample sugar chain and the internal standard sugar chain in the mass spectrum, such as comparing the peak area of the paired peak signal, the sample sugar chain peak area/internal Mark the ratio of sugar chain peak area (light/heavy) to obtain qualitative and/or quantitative information of sugar chain. It is also possible to combine the quality analysis data obtained by the method in this paper with the data obtained from other sugar chain analysis techniques for analysis.
  • mass spectrometry analysis due to the molecular weight difference between the sugar chains of the internal standard and the sugar chains of the sample, mass spectrometry analysis can show a specific difference in charge-to-mass ratio, distinguishable MS peak and peak area ratio. These data can be directly used for relative abundance comparison or qualitative analysis to infer the molecular structure of the target sugar chain; it can also be used to monitor the change in the abundance of the target sugar chain; or used to detect the presence, content and content of the substance with the target sugar chain. Dynamic changes.
  • sugar chain analysis software includes but is not limited to: Progenesis MALDI, GlycoWorkbench, NetNGlyc, FindMod, GlycanMass, GlycoMod, GlycoFragment, GlycoSearchMS, etc.
  • Available sugar chain databases include but are not limited to: GlycomeDB, EUROCarbDB, CarbBank, CCSD, etc.
  • the method herein can be used in high-throughput sugar chain detection, such as simultaneous detection of 48, 96, 192, 384 or more samples.
  • high-throughput sugar chain detection such as simultaneous detection of 48, 96, 192, 384 or more samples.
  • the method in this paper can be used for the qualitative and quantitative analysis of various sugar chains of interest in samples, and thus can be widely used in various applications related to sugar chain detection and analysis.
  • the method herein is used for the analysis of sugar chains related to physiological and pathological activities, such as sugar chains related to processes such as information transmission, cell growth and development, immune cell regulation, tumorigenesis and development.
  • the applicable aspects of the method herein include but are not limited to: for disease diagnosis based on sugar chain markers (such as cancer antigen 125 (CA125), carcinoembryonic antigen (CEA), prostate specific antigen (PSA)) and/or Prognostic judgment; used for screening potential disease-related sugar chain markers; used for the development and/or quality control of sugar complexes (such as sugar chain drugs, such as glycosylation modified antibodies); used for protein glycosylation Modification analysis; etc.
  • sugar chain markers such as cancer antigen 125 (CA125), carcinoembryonic antigen (CEA), prostate specific antigen (PSA)
  • Prognostic judgment used for screening potential disease-related sugar chain markers
  • sugar chain drugs such as glycosylation modified antibodies
  • protein glycosylation Modification analysis etc.
  • this article also provides a product used in the method and application herein, which contains a combination of reagents and/or equipment used in the method herein.
  • this application also uses the method of the present invention to analyze the difference in sugar groups in serum samples of lung cancer patients and healthy control serum samples, and found that 9 kinds of N-sugar chains (ie The part marked in gray in Table 3 of the embodiment section) can effectively distinguish lung cancer samples from healthy control samples (AUC>0.8).
  • these N-sugar chains alone or in combination can be used as markers for lung cancer diagnosis and/or lung cancer treatment plan screening:
  • N-glycan markers that can be used for lung cancer diagnosis and/or lung cancer treatment plan screening
  • H hexose
  • N N-acetylglucosamine
  • F fucose
  • E ⁇ 2,6-linked sialic acid
  • dark gray circle Man
  • light gray circle Gal
  • square GlcNAc
  • clockwise That is, the line is up
  • diamond ⁇ 2,6-connected sialic acid (ie E); counterclockwise (ie, the line is down)
  • the present disclosure also provides a product (such as a kit) for lung cancer diagnosis and/or lung cancer treatment plan screening, the product comprising: for detecting one of the above nine sugar chains in a sample Or multiple levels of substances (such as reagents and/or equipment); and optionally other substances used for lung cancer diagnosis, such as detection substances for existing lung cancer markers.
  • a product such as a kit
  • the product comprising: for detecting one of the above nine sugar chains in a sample Or multiple levels of substances (such as reagents and/or equipment); and optionally other substances used for lung cancer diagnosis, such as detection substances for existing lung cancer markers.
  • a method for diagnosing lung cancer and/or screening of treatment options for lung cancer comprising: determining the level of one or more of the above nine sugar chains in a sample obtained from a subject. Also disclosed herein is the application of substances that detect the level of one or more of the 9 sugar chains mentioned above in the preparation of products for cancer diagnosis and/or screening of lung cancer treatment options.
  • This article also provides a detection kit, which comprises: (i) a detection effective amount of one or more reagents for detecting one or more of the 9 sugar chain levels as described above; (ii) any Optionally, one or more substances selected from the following group: containers, instructions for use, positive controls, negative controls, buffers, adjuvants or solvents, such as solutions for suspending or fixing cells, detectable Labels or labels, solutions for lysing cells, reagents for releasing sugar chains, or reagents for sugar chain purification, etc.
  • an appropriate sugar chain detection substance can be selected and made into a product suitable for the detection method used.
  • Those of ordinary skill in the art can adjust and change the detection method and the substances contained in the product according to actual conditions and needs.
  • the biomimetic sugars described herein and related methods are preferably used to detect the level of the sugar chains in the sample.
  • the sample to be tested may be selected from: body fluid samples, such as blood, serum, plasma, urine, saliva, lymph, spinal fluid, ascites, and amniotic fluid; cell samples, such as cell samples isolated from tissues, Cell samples cultured in vitro; tissue samples, such as cancer tissues, para-cancerous tissues, and normal tissues, in the form of fresh tissue samples, immobilized tissue samples, etc.
  • body fluid samples such as blood, serum, plasma, urine, saliva, lymph, spinal fluid, ascites, and amniotic fluid
  • cell samples such as cell samples isolated from tissues, Cell samples cultured in vitro
  • tissue samples such as cancer tissues, para-cancerous tissues, and normal tissues, in the form of fresh tissue samples, immobilized tissue samples, etc.
  • the present application has the characteristics of high sensitivity and high accuracy in the diagnosis of lung cancer and/or the screening of lung cancer treatment plans by detecting the level of sugar chain markers.
  • the products and methods of the present application can also be used in combination with existing conventional lung cancer diagnosis methods, so that lung cancer can be diagnosed more sensitively and accurately. This combined use can produce a certain superposition or even additive effect.
  • Existing conventional lung cancer diagnosis methods include but are not limited to: computed tomography, circulating tumor cell (CTC) detection method (such as folate receptor positive CTC detection method), lung cancer autoantibody detection (such as P53, c-myc, HER2, NYESO -1, GAGE, MUG1 and GBU4-5 etc.).
  • glycoprotein standard IgG purchased from Sigma-Aldrich, product number 14506, the same below
  • normal saline 0.85% NaCl
  • PNGase F enzyme glycanase F
  • HILIC-SPE hydrophilic interaction chromatography-solid phase extraction
  • MQ ultrapure water
  • ACN 85% acetonitrile
  • 10 ⁇ L of ultrapure water was used to elute the reduced N-glycans.
  • sugar chain standard NA2G1F and 5 ⁇ L of glycoprotein standard IgG N-glycans were taken as samples, and 5 ⁇ L of reduced internal standard sugar chains were simultaneously esterified with ethanol (0.25M EDC and 0.25M HOBt dissolved in anhydrous Ethanol) derivatization, placed in a 37°C constant temperature and humidity incubator for 1 hour to protect the terminal sialic acid of the sugar chain.
  • HILIC-SPE hydrophilic interaction chromatography-solid phase extraction
  • sugar chain standard NA2G1F and the glycoprotein standard IgGN-sugar chain sample after the enzymolysis in step 2 are added to ultrapure water and diluted to the original concentration of 2, 5, 8, 10, 20, 50, 100 times.
  • mass calibration of the mass spectrometer is carried out with the mixed calibration solution TOFMix containing eight standard peptides.
  • the matrix super-DHB was dissolved in a 50% ACN solution containing 1 mM NaOH, with a final concentration of 5 mg/mL. Take 1 ⁇ L of the mixed sample and drop it on the mass spectrometer plate and dry it at room temperature; then add 1 ⁇ L of super-DHB matrix and dry it at room temperature; then add 0.2 ⁇ L of absolute ethanol to homogenize, so that the sample is evenly distributed on the target. Enhance the mass spectrum signal.
  • MALDI mass spectrometry collects signal ions in the positive ion reflection mode (reflection positive, RP) for signal ion detection.
  • RP positive ion reflection mode
  • the sample processing mode is "batch mode", which automatically controls the position of the laser point to reduce human operation errors.
  • the spectrum acquisition setting is: 2 shots/profile, and after averaging 200 profiles, one MS spectrum is collected, and the range of acquisition m/z is 1000-4000.
  • the end of the internal standard sugar chain is reduced to hydroxyl molecular weight + 2 Da, and the molecular weight of the isotope D label is +1 Da. Therefore, the final internal standard sugar chain molecular weight + 3 Da (as shown in Figure 1).
  • the ratio of the peak area of the sugar chain mass spectrum of the test sample/the peak area of the internal standard sugar chain mass spectrum (light/heavy) is obtained.
  • the content of each N-sugar chain is determined by the signal intensity ratio (light scale/heavy scale), which is obtained by calculating the peak area ratio of the highest isotope peak (sample/internal standard).
  • Each sample was spotted 3 times. According to the principle of collecting one MS spectrum for each target point for data analysis, the final data of each sample is the calculation result obtained after averaging 3 MS spectrum signals.
  • the reduction labeling efficiency can be investigated by comparing the mass spectra of the sugar chain standard NA2G1F (H4N4F1) before reduction labeling ( Figure 2 (A) upper panel) and after reduction labeling (2 (A) lower panel). As shown in the figure, the original m/z 1647.59 NA2G1F was not detected after reduction labeling, indicating that the reduction labeling efficiency of this method is close to 100%.
  • biomimetic sugar group prepared from the standard glycoprotein IgG N-sugar group covers a wide range of molecular weights, from m/z 1282.45 to 2653.93; the peak area coverage is also very wide, including 4 orders of magnitude, from 338 to 1082307.
  • the serum separation method is carried out according to the routine operation: first draw 5mL venous blood, leave it in a coagulation tube at room temperature for 30 minutes, centrifuge at 3000 rpm for 10 minutes after coagulation, aspirate the upper serum, and freeze it at -80°C for later use.
  • HILIC-SPE hydrophilic interaction chromatography-solid phase extraction
  • the N-sugar chain of the sample to be tested and the N-sugar chain of the internal standard were obtained through the above steps.
  • MALDI mass spectrometry collects signal ions in the positive ion reflection mode (reflection positive, RP) for signal ion detection.
  • the laser energy is set to 105-125V to minimize "in-source decay” (ISD) and improve the signal-to-noise ratio.
  • the sample processing mode is "batch mode", which automatically controls the position of the laser point to reduce human operation errors.
  • the spectrum acquisition setting is: 2 shots/profile, and after averaging 200 profiles, one MS spectrum is collected, and the range of acquisition m/z is 1000-4000.
  • the end of the internal standard sugar chain is reduced to hydroxyl molecular weight + 2 Da, and the molecular weight of the isotope D label is +1 Da. Therefore, the final internal standard sugar chain molecular weight + 3 Da (as shown in Figure 1).
  • the ratio of the peak area of the sugar chain mass spectrum of the sample to be tested/the peak area of the internal standard sugar chain mass spectrum (light/heavy) is obtained.
  • Each sample was spotted 3 times. According to the principle of collecting one MS spectrum for each target point for data analysis, the final data for each sample is the calculation result obtained after averaging 3 MS spectrum signals.
  • One serum sample was processed according to the internal standard process and stored in the refrigerator.
  • the other serum sample was divided into 3 parts, one of which was taken every day, and processed according to the sample process for 3 consecutive days. After mixing the internal standard and the samples processed for 3 consecutive days, the mass spectrometry analysis as described above was performed.
  • the average coefficient of variation CV of the 20 most abundant sugar chains is only 8.7%, which is also significantly lower than the currently available N-glycomic quantification method (CV: 16.5%)
  • CV N-glycomic quantification method
  • Lung cancer is one of the most common cancers in the world, and its 5-year survival rate is very low and very poor, only 8-16%. Therefore, there is an urgent need to find biomarkers with sensitivity and specificity for early lung cancer diagnosis. Changes in protein glycosylation have been reported to be closely related to the occurrence and development of lung cancer, and the identification and quantitative analysis of sugar chains have great potential for discovering related biomarkers.
  • N-sugar chains with significant statistical differences between lung cancer cases and healthy controls are listed in Table 3, including N-sugar chain structure, molecular weight, glycan/internal standard peak area ratio, p value and AUC.
  • H Hexose
  • N N-acetylglucosamine
  • F Iwasose
  • L ⁇ 2,3-linked sialic acid (lactonization)
  • E ⁇ 2,6-linked sialic acid (ethylation); composition The number in indicates the quantity;
  • the test results show that compared with healthy controls, there are 34 kinds of N-sugar chain expression levels increased in lung cancer serum samples.
  • the contents of degalactosylated N-sugar chains, fucosylated N-sugar chains, high-mannosylated N-sugar chains and multi-branched sialylated N-sugar chains were significantly increased in lung cancer samples.
  • H5N4L2 and H5N4E2 have the same glycan composition but different sialic acid linkages, which are obvious in distinguishing lung cancer from healthy controls.
  • the AUC is 0.72 and 0.91, respectively.
  • the method described herein can be used to effectively distinguish lung cancer from healthy subjects, and can be used to identify N-sugar chains with high AUC values as lung cancer tumor markers.
  • 9 sugar chains of H4N3, H3N3E1, H4N3E1, H5N4E1, H5N4E2, H5N5F1E1, H5N5E2, H6N5E2, and H6N5E3 can be used for effective and accurate diagnosis of lung cancer.

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Abstract

La présente invention concerne un sucre ou un groupe de sucre bionique marqué par un isotope, un procédé de préparation correspondant et une utilisation associée. Plus particulièrement, l'invention concerne un sucre ou un groupe de sucre bionique marqué par un isotope qui comprend un groupe hydroxyle réducteur d'alcool au niveau de l'extrémité réductrice d'une chaîne de sucre et d'un marqueur isotopique, par comparaison avec une chaîne de sucre non modifiée correspondante, chaque chaîne de sucre bionique a un poids moléculaire augmenté de 3 Daltons, et le sucre bionique (groupe) et le sucre non modifié (groupe) ont la même composition de chaîne de sucre et une distribution d'abondance de chaîne de sucre similaire. La présente invention concerne en outre un procédé d'analyse d'un groupe de sucre dans un échantillon par utilisation du sucre ou du groupe de sucre bionique marqué par un isotope, le procédé comprend la réalisation d'une analyse de masse d'un mélange de chaînes de sucre bionique standard interne marqué par un isotope et de chaînes de sucre d'échantillon non marqué par un isotope pour effectuer une analyse qualitative et/ou quantitative du groupe de sucre dans l'échantillon.
PCT/CN2019/130253 2019-04-24 2019-12-31 Sucre ou groupe de sucre bionique marqué par un isotope, procédé de préparation correspondant et utilisation associée WO2020215791A1 (fr)

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