WO2020215791A1

WO2020215791A1 - Isotope-labeled bionic sugar or sugar group, preparation method and application thereof

Info

Publication number: WO2020215791A1
Application number: PCT/CN2019/130253
Authority: WO
Inventors: 顾建新; 任士芳; 秦文俊
Original assignee: 复旦大学
Priority date: 2019-04-24
Filing date: 2019-12-31
Publication date: 2020-10-29
Also published as: CN110028539A; CN110028539B

Abstract

The present document discloses an isotope-labeled bionic sugar or sugar group, a preparation method and an application thereof Specifically, the isotope-labeled bionic sugar or sugar group disclosed in the present document comprises a reducing alcohol hydroxyl group at the reducing end of a sugar chain and an isotope label, wherein, compared to a corresponding unmodified sugar chain, each bionic sugar chain has a molecular weight increased by 3 Daltons, and the bionic sugar (group) and the unmodified sugar (group) have the same sugar chain composition and similar sugar chain abundance distribution. The present document further provides a method of analyzing a sugar group in a sample by using the isotope-labeled bionic sugar or sugar group, the method comprises performing a mass analysis of a mixture of isotope-labeled internal standard bionic sugar chains and non-isotope-labeled sample sugar chains to perform a qualitative and/or quantitative analysis of the sugar group in the sample.

Description

Isotope-labeled bionic sugar or sugar group, preparation method and application thereof

Technical field

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.

Background technique

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.

In recent years, considering the advantages of body fluid samples, people have begun to focus on the research of 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.

In addition, the qualitative and quantitative analysis of 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.

In recent years, with the improvement of the sensitivity of various analytical techniques, especially the development of biological mass spectrometry, quantitative glycomics has also developed rapidly. 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.

Although the 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.

Compared with the non-isotopic labeling quantitative method, the isotope labeling quantitative method is a more accurate quantitative method. By introducing stable isotope labels with the same chemical structure and similar physical properties but different mass numbers, the sugar chains in different samples are labeled, and then mixed for mass spectrometry detection and analysis. With 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. At present, 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.

However, current isotope labeling quantification methods each have their own shortcomings: enzymatically introduced isotope labels can only introduce 2Da molecular weight differences, which require additional deconvolution calculations; metabolic imported isotope labels are only applicable to cell samples, and the experimental cost is relatively high; However, most of the current methods of chemical derivatization to introduce isotope labeling are cumbersome. All samples need to undergo the same derivatization treatment, and the same group of samples usually need to be mixed together and then compared with another group of mixed samples. The samples are individually quantitatively analyzed.

In order to simplify these methods, researchers have developed some methods that add exogenous sugar chain standards such as isotope-labeled N-sugar chains, maltose series oligosaccharides, etc., to the samples as internal standards for quantitative analysis. And these methods proved to be effective for the quantitative analysis of N-sugar chains. However, due to the heterogeneity of natural glycan structure, wide molecular weight range and richness of abundance, it is ideal to provide an internal standard that has a sugar chain composition and abundance similar to the sugar group to be analyzed Characteristics, but this is still a challenge for now.

Therefore, there is an urgent need in the field to develop a simple, high-throughput, and accurate stable isotope internal standard sugar group analysis method that can completely cover all sugar chains in the sample to be tested, and has the same sugar chain composition and composition as the sample to be tested. Similar sugar chain abundance distribution, so that it can be used in research and development that require qualitative and quantitative analysis of protein glycosylation modification, such as screening for potential disease-related sugar chain markers and developing glycosylated antibody drugs Wait.

Summary of the invention

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 .

In one aspect, herein, there is provided 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.

In some embodiments, except for the reducing end modification, the biomimetic sugar has the same sugar chain composition and abundance as its corresponding unmodified sugar.

In some embodiments, the reducing end of the unmodified glycan is a hemiacetal group.

In some embodiments, the alcoholic hydroxyl group and the isotope-labeled reducing end of the unmodified glycan are produced by ring opening through a reduction reaction.

In some embodiments, 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:

On the other hand, in this article, a method for preparing an isotope-labeled biomimetic sugar or sugar group is provided, the method comprising:

(A) Provide sugar chains or sugar groups to be modified;

(B) Through a reduction reaction on the sugar chain or sugar group to be modified, the reducing end hemiacetal structure of the sugar chain or sugar group to be modified is converted into an alcoholic hydroxyl group and contains an isotope label,

Wherein, compared with the corresponding natural polysaccharide, the biomimetic sugar 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.

In some embodiments, the reduction reaction is performed using sodium borodeuteride (NaBD ₄ ).

In some embodiments, the sugar chain is an N-sugar chain or an O-sugar chain.

In some embodiments, the sugar chain to be modified includes one or more sugar chains.

In some embodiments, the sugar chains to be modified are obtained from natural samples.

In some embodiments, the sugar chain to be modified is obtained from a sample to be tested.

In some embodiments, 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.

In some embodiments, the sugar chain to be modified is a sugar chain released from a sugar complex.

In some embodiments, the sugar chain is released using PNGase F, Endoglycosidase H, F2, F3, Endoglycosidase II), chemical methods (such as β elimination reaction), and/or combinations thereof. .

In some embodiments, the method further includes protecting the sialic acid on the sugar chain, such as esterification protection.

On the other hand, in this article, an isotope-labeled biomimetic sugar or sugar group prepared by the method described above is provided.

On the other hand, in this article, a method for analyzing sugar chains or sugar groups in a sample is provided, and the method includes the following steps:

(i) Provide a sample sugar chain whose reducing end is hemiacetal;

(ii) Provide a 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;

(iii) mixing the sample sugar chain and the bionic sugar chain to form a mixture;

(iv) Perform quality analysis on the mixture;

(v) Based on the comparison and/or ratio of the mass analysis data of the sample sugar chain and the biomimetic sugar chain, qualitative and/or quantify the sample sugar chain.

In some embodiments, the biomimetic sugar is prepared using the method described herein.

In some embodiments, the method includes:

In 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; and/or

In 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/or

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.

In some embodiments, the sugar complex is selected from glycoproteins, proteoglycans, glycopeptides, glycolipids, or any combination thereof, such as sugar chain-containing antibodies.

In some embodiments, enzymatic methods (such as PNGase F, Endoglycosidase H, F2, F3, endoglycosidase II), chemical methods (such as β elimination reaction), and/or combinations thereof are used To release sugar chains.

In some embodiments, the purification and/or enrichment is performed by centrifugation, precipitation separation, filtration, chromatographic separation and the like.

In some embodiments, the comparison and/or ratio are obtained through calculation software and/or algorithms.

In some embodiments, each sugar chain that is not isotopically labeled in the sample has a corresponding isotope-labeled sugar chain.

In some embodiments, the method includes:

(a) Use PNGase F to digest the sugar chains on the sample glycoprotein, and optionally purify and/or enrich the sugar chains obtained;

(b) Reducing with NaBD ₄ and isotopically labeling part of the sugar chains obtained in (i) to obtain isotope-labeled sugar chains;

(c) Optionally, 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;

(d) Mix the isotope-labeled sugar chains and unlabeled sugar chains obtained in the previous step, and perform mass analysis of the resulting mixture, such as mass spectrometry analysis, such as matrix-assisted laser desorption ionization-quaternary ion trap-time of flight Mass spectrometry (MALDI-QIT-TOF MS) for analysis;

(e) By comparing the peak area of the paired peak signals in the mass spectrum, comparing the peak area of the sugar chain not labeled with isotope (such as the peak area of the mass spectrum) and the peak area of the sugar chain labeled with the isotope (such as the peak area of the mass spectrum). Relative quantitative.

In some embodiments, 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.

On the other hand, in this document, there is provided a product comprising the sugar chain or sugar group as described herein and/or reagents and/or equipment used in the method as described herein.

In some embodiments, 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.

On the other hand, in this article, lung cancer sugar chain markers selected from the following group are provided:

H4N3, H3N3E1, H4N3E1, H5N4E1, H5N4E2, H5N5F1E1, H5N5E2, H6N5E2, H6N5E3, or a combination of one or more of them;

Among them, H represents hexose, N represents N-acetylglucosamine, F represents fucose, and E represents α2,6-linked sialic acid.

On the other hand, in 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.

On the other hand, herein, there is provided a method for diagnosing lung cancer and/or screening for a treatment plan for lung cancer, the method comprising detecting the level of a lung cancer sugar chain marker as described above in a sample.

On the other hand, in this article, there is provided a kit 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.

In some embodiments, 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.

Those skilled in the art can arbitrarily combine the aforementioned technical solutions and technical features without departing from the inventive concept and protection scope of the present invention. Other aspects of the present invention are obvious to those skilled in the art due to the disclosure herein.

Description of the drawings

The present invention will be further described below in conjunction with the accompanying drawings, in which these displays are only to illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.

Figure 1: Schematic diagram of the flow of an embodiment of this application.

Figure 2: The mass spectrum of NA2G1F sugar chain:

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.

Figure 3: Mass spectrum of sugar chains on glycoprotein standard IgG:

Part A of Figure 3: mass spectrum of sugar chains without 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.

The glycosyl labels in the above figures are shown in Figure 1.

Detailed ways

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. In this method, since only the internal standard is labeled with reduced isotope, and the sample to be tested does not need to be labeled with isotope, it simplifies the sample processing process, saves time, reduces sample loss, and is more economical. In addition, a substance containing a mixture of sugar chains similar to the test sample (for example, obtained from the same or the same sample source) can be used as an internal standard, so that each sugar type in the test sample has a corresponding one. 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. Moreover, after adding the internal standard, only the mass spectrum peaks that appear in pairs in the mass spectrum (the molecular weight difference is 3Da or more, depending on the reduction and isotope labeling reagents used) are the sugar chains that need to be studied, and the others are impurities. This can eliminate the influence of non-sugar chains or non-target sugar chain interferences in samples (especially complex samples such as serum).

In addition, the method and the biomimetic sugar set described in this article have the advantages of simple operation, time saving, and lower experimental cost. We have successfully applied it to the quantitative analysis of sugar chains in complex biological samples (such as human serum). For example, 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. In summary, we have developed a novel relative quantitative method of glycomics and the corresponding biomimetic sugar panel, which has great potential in finding clinical biomarkers.

All numerical ranges provided herein are intended to clearly include all numerical values that fall between the end points of the ranges and numerical ranges between them. The features mentioned in the present invention or the features mentioned in the embodiments can be combined. All the features disclosed in this specification can be used in combination with any composition form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

As used herein, "containing", "having" or "including" includes "including", "consisting essentially of", "consisting essentially of", and "consisting of. ..... 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.

Steps before quality analysis

As used herein, the term "glycome" refers to all sugar chains expressed in a sample (such as cells, tissues) or all sugar chains on a specific type of glycoprotein.

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.

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.

Generally speaking of the latter, 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. In quantitative analysis, 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. In addition, 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.

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.

As used herein, the term "sugar chain reducing end" refers to the end of the glycan having a free hemiacetal hydroxyl group. In some embodiments, 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. For example, 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 β elimination reaction; also can use a combination of enzymatic and chemical methods to release sugar chains.

As described herein, in the method herein, only the internal standard sugar chains are isotopically labeled so that their molecular weight is higher than that of the unlabeled sample sugar chains, and the molecular weight difference can be at least 3 Da, such as 3 Da, 4 Da, 5 Da, and the like. In some embodiments, 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. In other embodiments, for O-sugar chains, NaBD ₄ can be used as a reducing reagent and H ₂ ¹⁸ O as a solvent when β 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.

After the sugar chains are subjected to any treatment, they can be purified and/or enriched using techniques known in the art. For example, 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.

Quality analysis and data processing

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.

In this method, the mass analysis of the mixture can be carried out in a suitable manner. These methods include but are not limited to: 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). It is preferable to use a technique that has a high degree of recognition of molecular weight differences for quality analysis, such as matrix-assisted laser desorption ionization mass spectrometry (MALDI MS).

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.

In 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.

Various sugar chain analysis software, applications, databases, algorithms, etc. can be used to analyze the obtained data. Available 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. When detecting a large number of samples, there is no need to perform reduction and isotope labeling of each sample, only the isotope labeling of the internal standard sugar chain, which greatly simplifies the sample preparation process, saves experimental costs, and reduces sample loss.

Applications and products

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.

In some embodiments, 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. For example, 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.

Correspondingly, 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.

Identification and application of sugar chain markers for lung cancer diagnosis and/or screening of lung cancer treatment options

As shown in the examples, 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). Thus, it is proved that these N-sugar chains alone or in combination can be used as markers for lung cancer diagnosis and/or lung cancer treatment plan screening:

Table 1. N-glycan markers that can be used for lung cancer diagnosis and/or lung cancer treatment plan screening

Among them, H=hexose, N=N-acetylglucosamine, F=fucose, E=α2,6-linked sialic acid; dark gray circle=Man; light gray circle=Gal; square=GlcNAc; clockwise ( That is, the line is up) diamond = α2,6-connected sialic acid (ie E); counterclockwise (ie, the line is down) diamond = α2,3-connected sialic acid (ie, L); triangle=Fuc.

Therefore, 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.

Correspondingly, a method for diagnosing lung cancer and/or screening of treatment options for lung cancer is also disclosed herein, the method 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.

According to the needs of the detection method used, 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. In some embodiments, the biomimetic sugars described herein and related methods are preferably used to detect the level of the sugar chains in the sample.

In some embodiments, 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.

In addition, 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. In addition, 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.).

Example

The present invention will be further described below in conjunction with specific embodiments. The following examples verify the reliability, effectiveness and practicability of the method of this application. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Those skilled in the art can make appropriate modifications and changes to the present invention, and these modifications and changes are within the scope of the present invention.

For the experimental methods that do not specify specific conditions in the following examples, conventional methods in the field can be used, for example, you can refer to the "Handbook of Glycomics" compiled by Richard D. Cummings et al. (Science Press, June 1, 2011) Or in accordance with the conditions recommended by the supplier.

Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the content described can be applied to the method of the present invention. The preferred implementation methods and materials described in this article are for demonstration purposes only.

Example 1. Investigation based on the linear relationship and coefficient of variation between sugar chain standards and glycoprotein standards

Take the N-glycan standard NA2G1F and glycoprotein standard IgG as examples for quantitative analysis to investigate the linear relationship of the quantitative analysis of this method, including the following steps:

1. Preparation of standard samples

Take 10μg of sugar chain standard NA2G1F (purchased from Ludger Company, the same below) and dissolve it in 200μL of ultrapure water to prepare a storage solution with a concentration of 0.05mg/mL.

Take 1 mg of glycoprotein standard IgG (purchased from Sigma-Aldrich, product number 14506, the same below) and dissolve it in 200 μL of normal saline (0.85% NaCl) to prepare a storage solution with a concentration of 5 mg/mL.

2. Enzymatic hydrolysis of glycoproteins

Take 20 μL glycoprotein IgG stock solution, add 40 μL 2% SDS solution, and place it in a 60°C constant temperature mixer for denaturation for 10 minutes. After denaturation, the temperature of the solution drops to room temperature, and 40 μL of enzymatic hydrolysis buffer solution (4% NP-40: 5×PBS=1:1, pH 7.5) is added, and shaken and mixed. Add 1 μL PNGase F enzyme (glycanase F, purchased from New England Biolabs, the same below), shake and mix, and incubate overnight (16-18h) in a constant temperature and humidity incubator at 37°C. Enzymatically hydrolyze the N-sugar chains on the glycoprotein standard IgG, and precipitate the protein with absolute ethanol. After centrifugation, the supernatant is aspirated to remove the precipitate.

3. Sodium borodeuteride NaBD ₄ reduces internal standard sugar chains

Take part of the sugar chain standard NA2G1F and the glycoprotein standard IgG N-sugar chain as internal standards, and add half the volume of the reaction system to the newly prepared 2M sodium borodeuteride NaBD ₄ (purchased from Sigma-Aldrich, dissolved in Ultrapure water), placed in a 65°C constant temperature mixer for 2 hours to reduce the internal standard sugar chain.

After the reduction is completed, HILIC-SPE (hydrophilic interaction chromatography-solid phase extraction) is used for enrichment and purification. Fill a 20μL pipette tip with cotton thread to make a purification cartridge. First, activate the column with 10μL of ultrapure water (MQ) 3 times; then, equilibrate the column with 10μL of 85% acetonitrile (ACN) 3 times; pipette the reduced system back and forth, and directly repeat the loading 40 times to ensure N -Sugar chains are completely adsorbed on the purification cartridge; then, wash the purification column with 10μL 85% ACN+1% trifluoroacetic acid (TFA) 3 times, and then wash the purification column 3 times with 10μL 85% ACN to remove salt and impurities ; Finally, 10μL of ultrapure water was used to elute the reduced N-glycans.

4. Ethanol esterification reaction

In addition, 5μL of sugar chain standard NA2G1F and 5μL of glycoprotein standard IgG N-glycans were taken as samples, and 5μL 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.

After the reaction, HILIC-SPE (hydrophilic interaction chromatography-solid phase extraction) was used for enrichment and purification. Fill a 20μL pipette tip with cotton thread to make a purification cartridge. First, activate the column with 10μL of ultrapure water (MQ) 3 times; then, equilibrate the column with 10μL of 85% acetonitrile (ACN) 3 times; pipette the reduced system back and forth, and directly repeat the loading 40 times to ensure The N-sugar chains are completely adsorbed on the purification cartridge; then, the purification column is washed 3 times with 10 μL 85% ACN+1% trifluoroacetic acid (TFA), and then the purification column is washed 3 times with 10 μL 85% ACN to remove salt and Impurities; Finally, 10μL of ultrapure water is used to elute the esterified N-sugar chains.

5. Mass spectrometry analysis

The 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.

Mix the above sugar chain standards NA2G1F and IgG N-sugar chain samples with different dilution multiples in equal volumes with their corresponding internal standard sugar chains (that is, the internal standard sugar chains reduced in step 3), and use matrix-assisted laser desorption ionization- Four-stage ion trap-time of flight mass spectrometry (MALDI-QIT-TOF MS) for analysis.

Before the analysis of the N-glycan sample, 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 1μL of the mixed sample and drop it on the mass spectrometer plate and dry it at room temperature; then add 1μL of super-DHB matrix and dry it at room temperature; then add 0.2μL 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. Use a 337nm nitrogen laser source and set the laser energy 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.

6. Data statistical analysis

Use Shimadzu Biotech MALDI MS and Progenesis MALDI software to process all the collected MALDI MS spectra, and then output to Microsoft Excel for analysis. The mass spectrum data is manually analyzed with the aid of GlycoWorkbench sugar chain analysis software. The identification of sugar chain structure is mainly based on the mass-to-charge ratio, the assignment of tandem mass spectrometry fragments, and previous N-glycomics related literature reports.

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). By comparing the peak area of the paired peak signals in the primary mass spectrum, 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.

7. Results and discussion

(1) Comparison of mass spectra of NA2G1F sugar chains with and without isotope reduction labeling is shown in Figure 2(A), and the mass spectrum of NA2G1F sugar chain mixture before and after isotope reduction labeling is shown in Figure 2(B). As shown in the figure, the end of the internal standard sugar chain is reduced to a 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, that is, migrates from m/z 1647.59 to m/z 1650.59.

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%.

(2) The investigation of the glycoprotein standard IgG containing a variety of complex N-glycans showed that: as shown in part A of Fig. 3 and part B of Fig. 3, after labeling by this method, IgG N-glycan Each signal has a corresponding biomimetic sugar signal with a difference of 3Da in molecular weight. The 24 hemiacetals detected in part A of Fig. 3 can be found in part B of Fig. 3 and the corresponding D-labeled alcohol sugars, whether they are neutral sugars (such as H3N4F1, m/z: 1485.53/ 1488.54) or acidic sugars (such as H5N4F1E1, m/z: 2128.53/2131.54) (as shown in the enlarged part in Figure 3), can be restored and labeled completely.

More importantly, at the same time, we also found that the abundance distribution of each pair of N-sugar chain peaks in part A of Figure 3 and part B of Figure 3 are similar. The 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.

(3) Analyze the peak area ratios of the N-glycan standard NA2G1F, H3N4F1 and H5N4F1E1 on the glycoprotein standard IgG with different dilutions and the internal standard. The results are shown in Figure 4 and Figure 5, respectively. The result shows that the stable isotope internal standard N-glycosome analysis method of the present application has a good linear relationship R ² ≥0.998 and a coefficient of variation of less than 15% in a dynamic range of two orders of magnitude.

This shows that this N-sugar chain quantification method shows good linearity in the range of two orders of magnitude (100 times) (dilution times), and the quantitative results have good stability.

Example 2. Sugar chain analysis in serum samples

In order to further verify the applicability of this quantitative method in complex biological samples, we verify the application of this quantitative method in the quantitative analysis of human serum N-glycosome through multiple repeated analysis experiments.

1. Serum sample collection and preservation

The blood samples were collected in the Cancer Hospital of Fudan University. All experimental operations and research contents have been approved by the Ethics Committee of Fudan University Affiliated Tumor Hospital, and written informed consent has been obtained from all subjects before sample collection.

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.

2. Enzymatic hydrolysis of sugar chains

Sample to be tested

Take 5 μL of serum sample, add 10 μL of 2% SDS, and denature at 60°C for 10 minutes. Then add 5 μL 4% NP-40, 5 μL 5×PBS and 1 μL PNGase F (glycanase F), and enzymatically digest at 37°C overnight (12-18h).

Internal standard

Take 20 μL of the above serum sample, add 40 μL of 2% SDS, and denature at 60°C for 10 minutes. Then add 20 μL 4% NP-40, 20 μL 5×PBS and 1 μL PNGase F, and enzymatically digest at 37°C overnight (12-18h).

3. Sodium borodeuteride NaBD ₄ reduces internal standard sugar chains

Take the internal standard enzymatic hydrolysis solution after enzymolysis, add absolute ethanol to precipitate the protein, centrifuge at high speed and draw the supernatant to remove the precipitate. Then, add a half volume of the newly prepared 2M sodium borodeuteride NaBD ₄ to the reaction system, and place it in a thermostatic mixer at 65°C for 2 hours.

4. Enrichment and purification of isotope-labeled internal standard sugar chains

After the reduction is completed, HILIC-SPE (hydrophilic interaction chromatography-solid phase extraction) is used for enrichment and purification. Fill a 20μL pipette tip with cotton thread to make a purification cartridge. First, activate the column 3 times with 10μL of ultrapure water (MQ); then, equilibrate the column with 10μL of 85% acetonitrile (ACN) 3 times; pipette the reduced system back and forth, and directly repeat the loading 40 times to ensure N -Sugar chains are completely adsorbed on the purification cartridge; then, wash the purification column with 10μL 85% ACN+1% trifluoroacetic acid (TFA) 3 times, and then wash the purification column 3 times with 10μL 85% ACN to remove salt and impurities ; Finally, 10μL of ultrapure water was used to elute the reduced N-glycans.

5. Ethanol esterification

Sample to be tested

Take 2μL of the serum sample solution after enzymolysis, add 20μL of derivatization reagents (0.25M EDC and 0.25M HOBt dissolved in absolute ethanol, react at 37°C for 60 minutes. Then use HILIC-SPE for enrichment and purification (the purification method is the same as 4).

Internal standard

Take 5μL of the internal standard N-sugar chain after reduction and purification, add 25μL of derivatization reagent (0.25M EDC and 0.25M HOBt dissolved in ethanol), and react at 37°C for 60 minutes. Then use HILIC-SPE for enrichment and purification (purification method is the same as 4).

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.

6. N-sugar chain mass spectrometry analysis of serum samples

Take 2μL of the N-sugar chain of the serum sample after enzymolysis, ethanol esterification, and purification, and mix with 4μL of the internal standard N-sugar chain after enzymolysis, reduction labeling, ethanol esterification, and purification, and then three points for each mixed sample Repeat spotting.

All mixed N-sugar samples were analyzed using matrix-assisted laser desorption ionization-quaternary ion trap-time of flight mass spectrometry (MALDI-QIT-TOF MS). Before the analysis of the N-glycan sample, 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 1μL of the mixed sample and drop it on the mass spectrometer plate and dry it at room temperature; then add 1μL of super-DHB matrix and dry it at room temperature; then add 0.2μL 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. Using a 337nm nitrogen laser source, 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.

7. Data statistical analysis

Use Shimadzu Biotech MALDI MS and Progenesis MALDI software to process all the collected MALDI MS spectra, and then output to Microsoft Excel for analysis. The mass spectrometry data is manually analyzed by GlycoWorkbench sugar chain analysis software. The identification of sugar chain structure is mainly based on the mass-to-charge ratio, the attribution of tandem mass spectrometry fragments, and previous N-glycomics reports (such as REIDING K R, BLANK D, KUIJPER D M, et al. High-Throughput Profiling of Protein N-Glycosylation by MALDI-TOF-MS Employing Linkage-Specific Serial Acid Esterification [J]. Analytical Chemistry, 2014, 86(12): 5784-93.).

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). By comparing the peak area of the paired peak signals in the primary mass spectrum, 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.

Example 3. Same day reproducibility investigation

On the same day, take a serum sample and process it as an internal standard according to the internal standard process, and take the same serum sample and divide it into 3 parts and process it according to the sample process. The internal standard serum sample processed according to the internal standard process was mixed with 3 samples, and then subjected to mass spectrometry analysis as described above.

After data analysis and processing, it can be seen that the average coefficient of variation CV of the 20 most abundant sugar chains is only 4.6%, which is significantly lower than the existing quantitative methods of N-glycomics (CV: 14.2%) (Vreeker, GCM; Nicolardi, S.; Bladergroen, MR; van der Plas, CJ; Mesker, WE; Tollenaar, R.; van der Burgt, YEM; Wuhrer, M., Automated Plasma Glycomics with Linkage-Specific Sialic Acid Esterification and UltrahighAnal Resolution MS 2018, 90(20), 11955-11961).

The results indicate that the quantitative method has excellent quantitative reproducibility in complex biological samples.

Example 4. Day-time reproducibility study

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.

According to data analysis and processing, 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%) (Vreeker, GCM; Nicolardi, S.; Bladergroen, MR; van der Plas, CJ; Mesker, WE; Tollenaar, R.; van der Burgt, YEM; Wuhrer, M., Automated Plasma Glycomics with Linkage-Specific Sialic Acid MSEsterification and Resolution Ultra high Anal Chem 2018, 90(20), 11955-11961).

Example 5. Quantitative glycomics study of lung cancer

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.

In order to further evaluate the quantitative analysis ability of our method in multiple complex biological samples, we used the quantitative method described in this article to analyze 32 human serum samples (including 16 lung cancer samples (cancer group) and 16 age-sex matched samples). Quantitative analysis was performed on healthy control samples (healthy control group).

Table 2. Basic data of lung cancer patients and healthy controls

Before the quantitative analysis of the N-sugar group, we used the BCA kit to measure the protein content in all serum samples and found that there was no statistical difference in the serum protein content between the cancer group and the healthy control group.

First, due to the biological diversity of different serum samples, we use mixed serum samples (that is, a mixture of serum from all lung cancer patients and healthy control samples) to prepare biomimetic sugar as an internal standard. Then, according to the above-mentioned experimental method, the internal standard N-sugar group and the N-sugar group of 32 samples were mixed separately, and the aforementioned MALDI-MS detection and analysis were performed. The detected N-sugar chains have a molecular weight difference of 3 Da for each pair, and all mass spectrum peaks are sodium peaks. A representative mass spectrum is shown in Figure 6.

According to the results of quantitative analysis, we compared 60 pairs of N-glycans with a CV of less than 25% between lung cancer and healthy controls. The 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.

Table 3. List of N-sugar chains with statistically different expression in lung cancer samples and healthy control samples

H=Hexose, N=N-acetylglucosamine, F=Iwasose, L=α2,3-linked sialic acid (lactonization), E=α2,6-linked sialic acid (ethylation); composition The number in indicates the quantity;

Dark gray circle=Man; light gray circle=Gal; square=GlcNAc; clockwise (that is, the line is up) diamond=α2,6-connected with sialic acid (that is, E); counterclockwise (that is, the line is down) rhombus=α2 , 3-linked sialic acid (ie L); triangle = Fuc

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. In previous studies, researchers used different quantitative analysis methods and found that the content of degalactosylated glycans in lung cancer serum samples increased.

According to the test results of the receiver operating characteristic curve (ROC), we found that 9 kinds of N-sugar chains (that is, the part marked in gray in Table 3) can effectively distinguish lung cancer samples from healthy control samples (AUC>0.8), including H4N3, H3N3E1, H4N3E1, H5N4E1, H5N4E2, H5N5F1E1, H5N5E2, H6N5E2 and H6N5E3 (H=hexose, N=N-acetylglucosamine, F=fucose, E=α2,6-linked sialic acid), these N-sugars Chain may be used as a potential marker for lung cancer diagnosis.

Interestingly, we also found that the changes of α2,6-linked sialic acid and α2,3-linked sialic acid were different in lung cancer samples. For example, H5N4L2 and H5N4E2 (L=α2,3-linked sialic acid, E=α2,6-linked sialic acid) have the same glycan composition but different sialic acid linkages, which are obvious in distinguishing lung cancer from healthy controls. For different abilities, the AUC is 0.72 and 0.91, respectively.

The above results indicate that 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. In addition, according to the identification results, 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.

All documents mentioned in the present invention are cited as references in this application, as if each document is individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

A modified isotope-labeled biomimetic sugar or a sugar group containing a modified isotope-labeled bionic sugar, wherein, compared with its corresponding unmodified glycan, the biomimetic sugar includes an alcohol hydroxyl group at the reducing end of the sugar chain and an isotope label, and The molecular weight of the bionic sugar has increased by 3 Daltons or more;

For example, the reducing end of the unmodified glycan is a hemiacetal group;

For example, the alcoholic hydroxyl group and the reducing end of the isotope-labeled unmodified glycan are generated by ring opening through a reduction reaction.
A method for preparing isotope-labeled bionic sugar or sugar group, the method comprising:

(A) Provide sugar chains or sugar groups to be modified;

(B) The reducing end hemiacetal structure of the sugar chain or sugar group to be modified is converted into an alcohol by a reduction reaction (for example, reduction with sodium borodeuteride or sodium borohydride) for the sugar chain or sugar group to be modified Hydroxyl and contains isotope labels,

Wherein, compared with the corresponding natural polysaccharide, the biomimetic sugar 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 isotope-labeled bionic sugar or sugar group prepared by the method of claim 2 is used.
A method for analyzing sugar chains or sugar groups in a sample, the method comprising the following steps:

(i) Provide a sample sugar chain whose reducing end is hemiacetal;

(ii) Provide a 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;

(iii) mixing the sample sugar chain and the bionic sugar chain to form a mixture;

(iv) Perform quality analysis on the mixture;

(v) Based on the comparison and/or ratio of the quality analysis data of the sample sugar chain and the bionic sugar chain, qualitatively and/or quantify the sample sugar chain;

Preferably, 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, cell samples cultured in vitro; Tissue samples, such as cancer tissues, adjacent tissues, and normal tissues, can be 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 sample;

Preferably, the sugar complex is selected from: glycoprotein, proteoglycan, glycopeptide, glycolipid, or any combination thereof, such as sugar chain-containing antibody, etc.;

Preferably, enzymatic methods (such as PNGase F, Endoglycosidase H, F2, F3, Endoglycosidase II), chemical methods (such as β elimination reaction) and/or combinations thereof are used to release sugar chains ；

Preferably, the purification and/or enrichment is performed by centrifugation, precipitation separation, filtration, chromatographic separation, etc.;

Preferably, the comparison and/or ratio are obtained through calculation software and/or algorithms; and/or

Preferably, each unlabeled sugar chain in the sample has an isotope-labeled sugar chain corresponding to it.
The method of claim 4, wherein the method comprises:

In 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; and/or

In 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/or

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 method of claim 4, wherein the method comprises:

(a) Use PNGase F to digest the sugar chains on the sample glycoprotein, and optionally purify and/or enrich the sugar chains obtained;

(b) Reducing with NaBD 4 and isotopically labeling part of the sugar chains obtained in (i) to obtain isotope-labeled sugar chains;

(c) Optionally, 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;

(d) Mix the isotope-labeled sugar chains and unlabeled sugar chains obtained in the previous step, and perform mass analysis of the resulting mixture, such as mass spectrometry analysis, such as matrix-assisted laser desorption ionization-quaternary ion trap-time of flight Mass spectrometry (MALDI-QIT-TOF MS) for analysis;

(e) By comparing the peak area of the paired peak signals in the mass spectrum, comparing the peak area of the sugar chain not labeled with isotope (such as the peak area of the mass spectrum) and the peak area of the sugar chain labeled with the isotope (such as the peak area of the mass spectrum). Relative quantitative.
The method of claim 4, wherein the method is further used for:

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.
A product comprising the sugar chain or sugar group according to claim 1 or 3 and/or reagents and/or equipment used in the method according to any one of claims 4-7.
The sugar chain or sugar group according to claim 1 or 3 and/or the reagent and/or equipment used in the method according to any one of claims 4-7 or the product according to claim 8 is in preparation Used for disease diagnosis and/or prognosis judgment based on sugar chain markers, screening of potential disease-related sugar chain markers, development of sugar complexes (such as sugar chain drugs, such as glycosylation-modified antibodies) Application in products for quality control and protein glycosylation modification analysis.
Application of lung cancer sugar chain markers selected from the following group and substances for detecting said lung cancer sugar chain markers in preparing products for lung cancer diagnosis and/or screening of lung cancer treatment plans:

H4N3, H3N3E1, H4N3E1, H5N4E1, H5N4E2, H5N5F1E1, H5N5E2, H6N5E2, H6N5E3, or any combination thereof;

Among them, H represents hexose, N represents N-acetylglucosamine, F represents fucose, and E represents α2,6-linked sialic acid.