Label for multi-channel biomolecule quantification and preparation method and application thereof

Abstract

The invention provides a label for multi-channel biomolecule quantification as well as a preparation method and application of the label. The label comprises a first channel label and a second channel label; wherein the raw materials of the first channel tag and the second channel tag are respectively selected from amino acid derivatives, and the amino acid derivatives comprise two or more amino acids which are isomers; the first channel label and the second channel label are same-quantity different-sequence labels. According to the label, the three-channel isomeride label and the two-channel isomeride label are coupled, so that the label has the isomeride, isomeride and isomeride characteristics, and compared with an existing tandem mass spectrum label technology, the label has the advantages of being simple in synthesis path, independent of high-resolution mass spectrum and label development potential and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry and mass spectrometry, and in particular, the present application relates to a tag for multi-channel biomolecule quantification, a preparation method and application thereof. BACKGROUND

[0002] There are various amino-containing biomolecules in organisms, including lipids, small molecule metabolites, proteins, etc. Taking phosphatidylethanolamine (PE) and phosphatidylserine (PS) as examples, both of which are amino glycerophospholipids with primary amine functional groups in the head group, in mammalian cells, PE and PS account for 20-50% and 2-10% of total membrane phospholipids, respectively. The distribution of these two phospholipids on the membrane presents a high degree of asymmetry, mainly enriched in the inner leaflet of the plasma membrane (cytoplasmic side). This specific localization is crucial for maintaining the structural integrity of the membrane (such as membrane curvature regulation) and mediating key cell signaling processes. In recent years, the biological functions of aminophospholipids have attracted increasing attention, because the transport of aminophospholipids across the plasma membrane is a key link in the process of cell activation, apoptosis, and aging, and is also essential for the removal of apoptotic cells and the coagulation process. In addition, abnormal metabolic regulation of aminophospholipid composition and distribution has been found to be closely related to the development of various human diseases, including cancer, diabetes, and neurodegenerative diseases. Therefore, accurate quantification and precise structural characterization of complex aminophospholipids are of great significance for understanding the metabolic pathways of aminophospholipids, revealing the pathological mechanisms of related diseases, and developing new therapeutic targets.

[0003] In modern proteomics, lipidomics, and metabolomics research, high-throughput quantitative analysis techniques based on mass spectrometry have become a key tool for analyzing the expression level changes of biomolecules in complex biological systems. Especially in the research of multi-sample parallel processing, disease marker screening, and biological pathway analysis, there are increasingly high requirements for high precision, multi-throughput, and quantitative accuracy.

[0004] Taking proteomics as an example, the current mainstream quantitative techniques mainly include the following categories: (1) Label-free quantification: by comparing the peak area or peak intensity of target peptides in different samples to estimate the relative abundance. This method is simple to operate, but is easily affected by sample processing, mass spectrometry condition fluctuations, etc. It takes a long time to operate the instrument, has relatively low reproducibility and accuracy, and lacks sample parallelism.

[0005] (2) Isotope internal standard method: a representative technology such as SILAC, by introducing peptides with known stable isotopes, relative quantification is achieved through light / heavy isotope reagent labeling by mass spectrometry, but its expansion ability is poor, the complexity of the spectrum is increased, the cost is high, the scope of application is limited, and it is also difficult to meet the needs of high-throughput sample analysis.

[0006] (3) Isobaric tags for relative and absolute quantitation (iTRAQ) and Tandem Mass Tag (TMT) are representative techniques. These techniques can realize parallel quantification of samples in the same mass spectrometry scan by applying tags with the same mass and different reporter ions. The advantages of these techniques are as follows: multiple sample quantification can be realized in a single experiment (which has been extended to 31 channels); sample processing is unified, reducing systematic errors; and these techniques are compatible with multiple mass spectrometry platforms. However, these techniques also have the following limitations: the tag design is complex, and the reagent is expensive (for example, the 6-channel TMT reagent of Thermo costs about 40390 yuan RMB per 4 mg); the resolution of reporter ions between different channels is limited, and signal overlap is prone to occur; and the channel expandability depends on the support of high-resolution mass spectrometers, increasing the equipment dependency.

[0007] In summary, isobaric tag technology is still the mainstream multi-channel quantification method at present. However, with higher requirements for quantification accuracy, channel expansion, and tag synthesis cost, traditional isobaric tags are facing new technical challenges. Therefore, there is an urgent need for a new type of mass spectrometry tag that can retain the advantages of isobaric tags while enhancing quantification accuracy, simplifying synthesis processes, and expanding more sample channels to achieve more efficient, low-cost, and accurate multi-channel biomolecule quantification analysis. SUMMARY

[0008] The present application aims to at least partially solve at least one of the technical problems existing in the prior art.

[0009] The present application is based on the following findings of the inventors: Although mass spectrometry-based quantification methods have been widely used in proteomics, lipidomics, and metabolomics research, existing high-throughput quantification methods still face many key challenges: traditional tags are expensive, isotopic internal standards are difficult to prepare, and the throughput is limited and the quantification error is large. Especially in complex biological samples, how to realize multi-channel synchronous quantification of target substances has become a technical bottleneck restricting the expansion of various types of omics research in depth and breadth.

[0010] Based on this, the present application proposes a new type of mass spectrometry tag with both isobaric and isomeric properties. The design idea is to couple a three-channel isomeric tag with a two-channel isobaric tag, and through the dual-label strategy of "structural difference + mass complementarity", both the bottleneck of limited channel number and single structure of traditional TMT are broken through, and the quantification throughput and isomeric recognition ability are significantly improved. Compared with the existing TMT technology, the tag of the present application has the following advantages: 1) High-throughput scalability - the number of channels can be extended with synthetic modularization; 2) Low cost - simple synthetic route, no need for expensive isotopic raw materials; 3) Wide range of applications - can be covalently combined with any biological molecule containing amino functional groups (such as lipids, peptide segments); 4) Low equipment requirements - quantitative does not depend on high-resolution mass spectrometry; 5) Great potential for iteration - the molecular skeleton is easy to derivatize, facilitating subsequent upgrades.

[0011] Therefore, in the first aspect of the present application, the present application proposes a tag for multi-channel biomolecule quantification, characterized in that it comprises: a first channel tag and a second channel tag; wherein the raw materials of the first channel tag and the second channel tag are selected from two or more amino acid derivatives, and the amino acid derivatives include two or more isomers of amino acids; the first channel tag and the second channel tag are isobaric tags. Thus, the present application uses amino acid derivatives as the skeleton, couples isomer tags (3 channels) and isobaric tags (2 channels) into a "double-tag" system, realizes 6-channel parallel quantification, and can be further expanded to 63 channels, breaking through the limitations of traditional isobaric tags in terms of limited number of channels and single structure, improving the quantification throughput while enhancing the isomer recognition ability; at the same time, the tag of the present application is low in cost and has a wide range of applications.

[0012] In the second aspect of the present application, a method for preparing the tag of the first aspect is proposed. According to an embodiment of the present application, the method comprises: performing a reduction methylation reaction on part of the amino acid derivatives to obtain a first channel tag or a second channel tag. Thus, by the method of the present application, the balance group can "make up" the inherent mass difference between different reporter groups by precise matching of isotopes, ensuring that all six-channel tags are completely consistent in overall molecular mass. This feature makes the tag appear as a single mass spectrometry peak in the first mass spectrometry, achieving the core goal of isobaric entry of multi-channel samples into the mass spectrometry system; and after fragmentation, different channel tags release reporter ions with characteristic mass differences, thereby completing multi-channel differentiation and laying a precise foundation for subsequent quantitative analysis. In addition, the method of the present application is efficient and convenient to synthesize, does not require complex processes, can meet large-scale production needs, and the tag yield can reach 70%, which can greatly reduce the cost, with a cost of only 30 yuan per 5 mg.

[0013] In a third aspect, the present application provides a kit comprising the tag of the first aspect or the tag prepared by the method of the second aspect. As described above, the tag of the present application is based on the coupling design of three-channel isomer tags and two-channel isobaric labels, so that the kit of the present application can directly realize 6-way sample parallel quantification by ion mobility (IM) separation technology, and can realize accurate identification and quantification of isomers without ultra-high resolution mass spectrometry, reduce the dependence on expensive instruments, have a wider application range, and can be used for biomolecules such as reactants containing amino groups, fatty acids, sugars, phospholipids, and sulfuric acid lipids.

[0014] In a fourth aspect, the present application provides a method for derivatizing biomolecules. According to an embodiment of the present application, the method comprises: derivatizing the biomolecules by using the tag of the first aspect, the tag prepared by the method of the second aspect, or the kit of the third aspect, to obtain derivatized biomolecules. It is found by experiments that the biomolecules are derivatized by using the tag of the present application, which can efficiently realize labeling of target biomolecules and reduce interference of unreacted substrates. At the same time, after derivatization, the chromatographic peak intensity of biomolecules (such as amino phospholipids) of the same concentration is increased by 2-3 times compared with that before derivatization, which enhances the detection sensitivity and is beneficial to qualitative and quantitative analysis of low-abundance target molecules. In addition, the tag has isomerism and isobarism characteristics, and after derivatization, ion mobility separation and tandem mass spectrometry detection can be realized to achieve six-way sample parallel quantification, which significantly improves the throughput of complex sample analysis.

[0015] In a fifth aspect, the present application provides a method for multi-channel quantification of biomolecules. According to an embodiment of the present application, the method comprises: derivatizing the biomolecules by using the method of the fourth aspect to obtain derivatized biomolecules; and analyzing the derivatized biomolecules to determine the content of the biomolecules. Thus, the method of the present application can directly realize six-way sample parallel quantification detection, greatly improve the throughput of complex sample analysis in the fields of proteomics and lipidomics, and meet the demand for parallel processing of multiple samples. In addition, the tag realizes isobarism by adjusting the mass of the balancing group, and can effectively distinguish isomers, reduce signal overlap interference, and accurately reflect the actual proportion of target biomolecules by combining ion mobility separation and tandem mass spectrometry technology.

[0016] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter in the description of embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0017] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description of embodiments, taken in conjunction with the accompanying drawings. Figure 1For isomer-homologues co-use six-channel tag structure according to the embodiment of the present application, its structure is composed of reporter group (red part), balance group (blue part) and reaction group (black part). Indicate the tag with 3-amino-4, 4-dimethylvaleric acid as the skeleton by triangle symbol, indicate the tag with 2-aminoheptanoic acid as the skeleton by square symbol, and indicate the tag with 2-amino-4, 4-dimethylvaleric acid as the skeleton by round symbol. d2 / d4 represents the homologues tag of two channels; Figure 2 is a nuclear magnetic spectrum of isomer-homologues co-use six-channel tag according to the embodiment of the present application; wherein, Figure 2a For the reporter ion with 2-(dimethylamino) heptanoic acid as the skeleton m / z For the tag of 130.123 channel; Figure 2b For the reporter ion with 2-(dimethylamino) heptanoic acid as the skeleton m / z For the tag of 132.143 channel; Figure 2c For the reporter ion with 2-(dimethylamino)-4, 4-dimethylvaleric acid as the skeleton m / z For the tag of 130.123 channel; Figure 2d For the reporter ion with 2-(dimethylamino)-4, 4-dimethylvaleric acid as the skeleton m / z For the tag of 132.143 channel; Figure 2e For the reporter ion with 3-(dimethylamino)-4, 4-dimethylvaleric acid as the skeleton m / z For the tag of 116.110 channel; Figure 2f For the reporter ion with 3-(dimethylamino)-4, 4-dimethylvaleric acid as the skeleton m / z For the tag of 118.123 channel; Figure 3 Figure 1 is a lipid derivatization process and UPLC chromatogram according to the embodiment of the present application; wherein, Figure 3 a is a specific process of derivatizing lipids by using 2-amino-4, 4-dimethylvaleric acid as the skeleton; Figure 3 b is the chromatogram before and after derivatization of phosphatidylethanolamine PE 16:0_18:1 / phosphatidylserine PS 16:0_18:1, the concentration of the lipids after derivatization is 8 μg / mL, and the injection amount is 3 μL; Figure 4 Figure 2 is a nuclear magnetic spectrum of isomer-homologues co-use six-channel tag according to the embodiment of the present application; wherein, Figure 4 a, d are the mass spectra of phosphatidylethanolamine PE 16:0_18:1 / phosphatidylserine PS 16:0_18:1 after derivatization; Figure 4 b, e are the secondary mass spectra of phosphatidylethanolamine PE 16:0_18:1 / phosphatidylserine PS 16:0_18:1 before derivatization; Figure 4 c, f are the secondary mass spectra of phosphatidylethanolamine PE 16:0_18:1 / phosphatidylserine PS 16:0_18:1 after derivatization; Figure 5 isomers tag reagent according to the embodiment of the present application for analyzing phosphatidylethanolamine PE 16:0_18:1; wherein, Figure 5 a is the chromatogram before and after derivatization of phosphatidylethanolamine PE 16:0_18:1 with 2-amino-4,4-dimethylpentanoic acid as the backbone tag; Figure 5 b is the mass spectrum of derivatized phosphatidylethanolamine PE 16:0_18:1 via CID secondary fragmentation; Figure 5 c is the extracted ion mobility plot of three isomer tag derivatization product fragments after cyclic IMS mobility separation; Figure 5 d is the TAP CID fragmentation of the fragment of 297.1 using the time alignment parallel fragmentation function specific to cyclic IMS to obtain the tertiary mass spectra of different isomer tags; m / z Figure 6 is the flow of phosphatidylethanolamine PE 16:0_18:1 labeled with 6-channel isomer-isobaric labeling according to the embodiment of the present application; wherein the mixed concentration ratios of 3-(dimethylamino)-4,4-dimethylpentanoic acid (triangle) d2 / d4, 2-(dimethylamino)-4,4-dimethylpentanoic acid (circle) d2 / d4 and 2-(dimethylamino) heptanoic acid (square) d2 / d4 are 1:1, 1:2 and 1:4 respectively; the mixed sample is subjected to RPLC-MS 2 CID to produce lipid head group fragments carrying isomer tags, and separated by 6 ion mobility circles, and finally by TAP MS 3 -CID to obtain each channel reporter ion; Figure 7 is the box plot of the 1:1:1:1:1:1 labeled lipid mixture reporter ion ratio and the two 1:1 unlabeled lipid ion intensity ratios according to the embodiment of the present application; Ch130 and Ch132 are two isobaric tag reporter ions​m / z Value; the 1st-2nd box plot corresponds to the reporter ion with 2-aminoheptanoic acid as the isomer tag, the 3rd-4th box plot corresponds to the reporter ion with 2-amino-4,4-dimethylpentanoic acid as the isomer tag, the 5th-6th box plot corresponds to the reporter ion with 2-amino-4,4-dimethylpentanoic acid as the isomer tag, and the 7th-8th box plot corresponds to the unlabeled lipid Figure 8 Figure 1 is a lipid quantification plot according to an embodiment of the present application; wherein, Figure 8 a is a quantification plot obtained by the relationship between the theoretical value and the experimental value after the phosphatidylethanolamine PE 16:0_18:1 is derivatized with three isomer tags, and the ratio of the two isobaric tags is increased from 10%-30%-50%-70%-90%; Figure 8 b is a quantification plot obtained by the relationship between the theoretical value and the experimental value after the phosphatidylserine PS 16:0_18:1 is derivatized with three isomer tags, and the ratio of the two isobaric tags is increased from 10%-30%-50%-70%-90%; Figure 9 Figure 2 is an experimental flow chart for preparing the tags according to an embodiment of the present application. DETAILED DESCRIPTION

[0018] The embodiments of the present application are described in detail below, which are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0019] Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are they necessarily mutually exclusive of one another. It is explicitly contemplated that embodiments described herein can be combined with other embodiments.

[0020] For the sake of brevity, only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form a range not explicitly recited; and any lower limit can be combined with any other lower limit to form a range not explicitly recited, and likewise any upper limit can be combined with any other upper limit to form a range not explicitly recited. Further, each individual disclosed point or single numerical value can be combined with any other point or single numerical value to form a range not explicitly recited, either as a lower limit or an upper limit, or in combination with other lower limits or upper limits.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this application; the use of the terms "including," "comprising," or "having" and variations thereof herein is intended to be broad and encompass the terms "consisting of" and "consisting essentially of" and variations thereof. Unless otherwise required by context, singular terms shall include pluralities and vice versa. Unless otherwise required by context, the use herein of the singular is also to be construed as a generic reference to the plural and vice versa.

[0022] In this document, the terms "comprise" or "comprising" are open- ended, that is, they mean "including, but not limited to".

[0023] It has to be noted that, as used herein, the terms "first", "second" and the like do not imply any particular order, but are used to distinguish one element from another. Further, it is to be understood that the terms "comprise", "comprising", "comprises" and / or "comprising" when used in this specification are taken to specify the presence of stated features, integers, steps and / or components but do not preclude the presence or addition of one or more other features, integers, steps, components and / or groups thereof.

[0024] The term "biomolecule" is not particularly limited and any type of molecule can be provided for testing using the method of the present application, which is amenable to mass spectrometric analysis and can be labeled with isobaric tags for mass spectrometry comprising different mass label groups. Biomolecules include amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleic acids, polynucleotides, oligonucleotides, DNA, RNA, peptide-nucleic acids, sugars, starches and complex carbohydrates, fats and complex lipids, polymers and small organic molecules, such as drugs and drug-like molecules or fragments thereof. Biomolecules are preferably lipids, amino acids, peptides, polypeptides, proteins. For the present application, the term analyte is synonymous with the term biomolecule.

[0025] The term "protein" shall include any molecule comprising two or more amino acids, including dipeptides, tripeptides, peptides, polypeptides and proteins.

[0026] The term "reporter group" is the key part of the tag for realizing the multi-channel distinction, which contains different skeleton structures and isotope labels. In tandem mass spectrometry detection, the group will release reporter ions with characteristic mass after fragmentation, and by identifying these different mass reporter ions, the samples of different channels can be distinguished, providing the basis for quantitative analysis. For example, the tags with 3-amino-4,4-dimethylpentanoic acid, 2-amino-4,4-dimethylpentanoic acid, 2-aminoheptanoic acid as the skeleton, the reporter groups will produce different reporter ions due to the differences in skeleton and isotope labeling. m / z

[0027] ​The term "balancing group" is mainly used for mass adjustment to achieve isobaric tags. Through precise isotope combination, the balancing group can "make up" the mass difference between different reporter groups, ensuring that the overall molecular mass of the tags in all channels is the same, so that they appear as the same peak in the first mass spectrum, meeting the requirement of "isobaric entry into mass spectrum" and laying the foundation for subsequent multi-channel parallel analysis.

[0028] The term "reactive group" is used to selectively covalently link to target biomolecules (such as amino-containing functional groups), so as to label the target molecules with tags. This specific reaction ensures that the tags are only combined with amino-containing biomolecules, ensuring the specificity and effectiveness of derivatization, and is the prerequisite for quantitative analysis of target molecules.

[0029] In the field of organic chemistry, the term "isomer" refers to those compounds with the same molecular formula but completely different atomic arrangement and structure, which is called isomerism. Isomers mainly include constitutional isomers and stereoisomers.

[0030] The term "isobaric tags" is a labeling technology for biomolecular quantitative analysis. By adding tags with the same mass to different samples, different reporter ions can be released after fragmentation, achieving the purpose of parallel quantitative analysis of multiple samples in the same mass spectrum scan. In common isobaric tag quantitative technologies such as TMT (tandem mass spectrometry tag) and iTRAQ (relative and absolute quantitative isobaric tag), the technology has obvious advantages, can realize quantitative analysis of multiple samples in a single experiment, and the sample processing method is unified, which can reduce systematic error and is compatible with multiple mass spectrometry platforms. However, it also has some defects, such as complex tag design structure, leading to high reagent price, limited resolution of reporter ions between different channels, easy signal overlap, and channel expansion dependent on high-resolution mass spectrometers.

[0031] The present application provides a tag for multi-channel biomolecular quantification, a method for preparing the tag, a kit, a method for derivatizing biomolecules, and a method for multi-channel quantification of biomolecules, which will be described in detail below.

[0032] Tag for multi-channel biomolecular quantification In a first aspect, the present application provides a label for multi-channel biomolecule quantification, characterized in that it comprises: a first channel label and a second channel label; wherein the raw materials of the first channel label and the second channel label are selected from two or more amino acid derivatives, the amino acid derivatives including two or more isomers of amino acids; and the first channel label and the second channel label are isobaric labels. Thus, the present application uses amino acid derivatives as the backbone, couples isomer labels (3 channels) and isobaric labels (2 channels) into a "double label" system, realizes 6-channel parallel quantification, and can be further expanded to 63 channels, breaking through the limitations of traditional isobaric labels, such as limited channel number and single structure, improving the quantification flux while enhancing the isomer recognition ability; at the same time, the label of the present application has low cost and wide application range.

[0033] In some embodiments of the present application, the raw materials of the first channel label and the second channel label are the same. In some embodiments of the present application, the amino acid derivatives include 3-amino-4,4-dimethylpentanoic acid, 2-amino-4,4-dimethylpentanoic acid, and 2-aminoheptanoic acid. Thus, by using the same raw material for the two channel labels, the isobaric characteristics can be realized through the difference in isotopic labeling. That is, by introducing different isotopic labels later, the two channel labels remain consistent in overall molecular mass. This ensures that the labeled molecules of the two channels appear as the same peak in the first mass spectrum and can be synchronized into the subsequent analysis process, meeting the core requirement of "isobaric entry into mass spectrometry".

[0034] In some embodiments of the present application, the first channel label comprises a first reporter group, a first equilibrium group, and a first reactive group. In some embodiments of the present application, the first reporter group is selected from at least one of 、 、 In some embodiments of the present application, the first equilibrium group is selected from =O. In some embodiments of the present application, the first reactive group is selected from -COOH.

[0035] In some embodiments of the present application, the second channel label comprises a second reporter group, a second equilibrium group, and a second reactive group. In some embodiments of the present application, the second reporter group is selected from at least one of 、 、 In some embodiments of the present application, the second equilibrium group is selected from =O. In some embodiments of the present application, the second reactive group is selected from -COOH. 18 O. In some embodiments of the present application, the second reactive group is selected from -CO 18 O 18 OH.

[0036] In some embodiments of the application, the first channel label is selected from at least one of the following: 、 、 .

[0037] In some embodiments of the application, the second channel label is selected from at least one of the following: 、 、 .

[0038] Method for preparing a label In a second aspect, the present application provides a method for preparing the label of the first aspect. According to embodiments of the present application, as shown in Figure 9 the method comprises: S100: isotopic labeling In the process, part of the amino acid derivative is isotopically labeled to obtain an isotopically labeled amino acid.

[0039] In some embodiments of the application, the isotopic labeling is performed by mixing the amino acid derivative with an isotopic solution containing trifluoroacetic acid to obtain a mixture; the mixture is subjected to a first reaction treatment to obtain a first reaction product; the trifluoroacetic acid in the first reaction product is removed to obtain a second reaction product; the second reaction product is sequentially subjected to pH adjustment, precipitation, and washing to obtain the isotopically labeled amino acid.

[0040] In some embodiments of the application, the mass-to-volume ratio of the amino acid derivative to the isotopic solution containing trifluoroacetic acid is 100 mg:(1-5) mL. For example, it can be 100 mg:1 mL, 100 mg:2 mL, 100 mg:3 mL, 100 mg:4 mL, 100 mg:5 mL, etc., or it can be a range consisting of any of the above values.

[0041] In some embodiments of the application, the volume ratio of trifluoroacetic acid in the isotopic solution containing trifluoroacetic acid is 5%-10%. For example, it can be 5%, 6%, 7%, 8%, 9%, 10%, etc., or it can be a range consisting of any of the above values.

[0042] In some embodiments of the application, the first reaction treatment is performed at 60-70°C. For example, it can be 60°C, 62°C, 64°C, 65°C, 66°C, 68°C, 70°C, etc., or it can be a range consisting of any of the above values.

[0043] In some embodiments of the present application, the first reaction treatment is performed for 15-25 hours. For example, it can be 15 hours, 17 hours, 19 hours, 20 hours, 23 hours, 25 hours, etc., or it can be a range consisting of any of the above values.

[0044] In some embodiments of the present application, the pH is adjusted to 5.5-6.5. For example, it can be 5.5, 5.7, 5.9, 6, 6.2, 6.5, etc., or it can be a range consisting of any of the above values. In some embodiments of the present application, the pH adjustment is performed using an ammonium bicarbonate solution.

[0045] In some embodiments of the present application, the precipitation is performed using cold diethyl ether. In some embodiments of the present application, the washing is performed using cold diethyl ether.

[0046] S200: Reductive methylation reaction In this process, the amino acid derivative or the isotopically labeled amino acid is subjected to a reductive methylation reaction to obtain a first channel tag or a second channel tag.

[0047] In some embodiments of the present application, the amino acid derivative comprises at least one of 3-amino-4,4-dimethylpentanoic acid, 2-amino-4,4-dimethylpentanoic acid, and 2-aminoheptanoic acid.

[0048] In some embodiments of the present application, the reductive methylation reaction is performed in the presence of a reactant and a reducing agent. In some embodiments of the present application, the reducing agent is selected from sodium cyanoborohydride or sodium cyanoborodeuteride. In some embodiments of the present application, the reactant is selected from formaldehyde or deuterated formaldehyde.

[0049] In some embodiments of the present application, the molar ratio of the amino acid derivative or the isotopically labeled amino acid, the reactant, and the reducing agent is 1:(2-3):(2-3). For example, it can be 1:2:2, 1:2.2:2.2, 1:2.4:2.4, 1:2.5:2.5, 1:2.7:2.7, 1:3:3, 1:2:3, 1:3:2, etc., or it can be a range consisting of any of the above values.

[0050] In some embodiments of the present application, the reductive methylation reaction is performed on the amino acid derivative, the reducing agent is selected from sodium cyanoborohydride, and the reactant is selected from deuterated formaldehyde. In some embodiments of the present application, the reductive methylation reaction is performed on the isotopically labeled amino acid, the reducing agent is selected from sodium cyanoborodeuteride, and the reactant is selected from formaldehyde.

[0051] In some embodiments of the present application, after the methylation reaction, the product of the methylation reaction is further subjected to freeze-drying treatment, and the product after the freeze-drying treatment is subjected to purification treatment to obtain the first channel tag or the second channel tag. In some embodiments of the present application, the purification treatment is performed by column chromatography. In some embodiments of the present application, the mobile phase of the column chromatography is a mixture of methanol and dichloroethane.

[0052] Kit In a third aspect of the present application, a kit is provided, which comprises the tag of the first aspect or the tag prepared by the method of the second aspect. As described above, the tag of the present application is based on the coupling of a tri-channel isomer tag and a two-channel isomer label, so that the kit of the present application can realize 6-fold sample parallel quantification directly by ion mobility (IM) separation technology, and the isomer can be accurately identified and quantified without the need of ultra-high resolution mass spectrometry, which reduces the dependence on expensive instruments, has a wider application range, and can be used for biological molecules such as reactants containing amino groups, fatty acids, sugars, phospholipids, and sulfuric acid lipids.

[0053] Method In a fourth aspect of the present application, a method for derivatizing a biological molecule is provided. According to embodiments of the present application, the method comprises: derivatizing the biological molecule by using the tag of the first aspect, the tag prepared by the method of the second aspect, or the kit of the third aspect to obtain a derivatized biological molecule. It is found by experiments that the biological molecule is derivatized by using the tag of the present application, which can efficiently realize the labeling of the target biological molecule and reduce the interference of unreacted substrates. At the same time, after derivatization, the chromatographic peak intensity of the biological molecule (such as amino phospholipid) of the same concentration is increased by 2-3 times compared with that before derivatization, which enhances the detection sensitivity and is beneficial to the qualitative and quantitative analysis of low-abundance target molecules. In addition, the tag has the characteristics of isomerism and isomerism, and after derivatization, it can be detected by ion mobility separation and tandem mass spectrometry to realize six-fold sample parallel quantification, which significantly improves the throughput of complex sample analysis.

[0054] In some embodiments of the present application, the method comprises: respectively activating the first channel label and / or the second channel label in the label or the first channel label and / or the second channel label in the kit to obtain a first activated label and a second activated label; respectively mixing the first activated label and / or the second activated label with the biomolecule to obtain a first mixed product and / or a second mixed product; respectively performing a second reaction treatment on the first mixed product and / or the second mixed product to obtain a third reaction product and / or a fourth reaction product; respectively mixing the third reaction product and / or the fourth reaction product with a mixed solution containing chloroform to obtain a third mixed product and / or a fourth mixed product; performing centrifugal treatment on the third mixed product and / or the fourth mixed product to collect a chloroform layer solution; and blowing dry the chloroform layer solution to obtain the derivatized biomolecule. Thus, by the method of the present application, the activated label can be specifically combined with the biomolecule, so that the biomolecules of different samples carry labels (containing isomers and isobaric markers) with distinguishing characteristics, and by combining liquid chromatography, ion mobility and tandem mass spectrometry, parallel qualitative and quantitative detection of six samples can be realized, and the analysis throughput of complex samples is improved.

[0055] In some embodiments of the present application, the method comprises: activating the first channel label to obtain a first activated label; mixing the first activated label with the biomolecule to obtain a first mixed product; performing a second reaction treatment on the first mixed product to obtain a third reaction product; mixing the third reaction product with a mixed solution containing chloroform to obtain a third mixed product; performing centrifugal treatment on the third mixed product to collect a chloroform layer solution; and blowing dry the chloroform layer solution to obtain the biomolecule derivatized by the first channel label.

[0056] In some embodiments of the present application, the method comprises: activating the second channel label to obtain a second activated label; mixing the second activated label with the biomolecule to obtain a second mixed product; performing a second reaction treatment on the second mixed product to obtain a fourth reaction product; mixing the fourth reaction product with a mixed solution containing chloroform to obtain a fourth mixed product; performing centrifugal treatment on the fourth mixed product to collect a chloroform layer solution; and blowing dry the chloroform layer solution to obtain the biomolecule derivatized by the second channel label.

[0057] In some embodiments of the present application, the activation treatment is performed in the presence of a catalyst. In some embodiments of the present application, the catalyst comprises 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride, 1-hydroxybenzotriazole, N-methylmorpholine.

[0058] In some embodiments of the present application, the molar ratio of the first channel tag or the second channel tag, the 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride, the 1-hydroxybenzotriazole and the N-methylmorpholine is 1:(0.5-1):(0.1-0.5):(0.5-1). For example, it can be 1:0.5:0.1:0.5, 1:0.7:0.3:0.7, 1:0.9:0.4:0.9, 1:1:0.5:1, or a range consisting of any of the above values.

[0059] In some embodiments of the present application, before the biomolecule is subjected to the first mixing treatment, the biomolecule is further dissolved in anhydrous N,N-dimethylformamide containing triethylammonium bicarbonate buffer.

[0060] In some embodiments of the present application, the molar ratio of the biomolecule to the first channel tag or the second channel tag is 1:(500-1000). For example, it can be 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, or a range consisting of any of the above values.

[0061] In some embodiments of the present application, the second reaction treatment is performed at a temperature of 55-65°C. For example, it can be 55°C, 57°C, 59°C, 60°C, 62°C, 65°C, or a range consisting of any of the above values.

[0062] In some embodiments of the present application, the second reaction treatment is performed for a time period of 0.5-1.5h. For example, it can be 0.5h, 0.7h, 0.9h, 0.1h, 1.2h, 1.5h, or a range consisting of any of the above values.

[0063] In some embodiments of the present application, the mixture containing chloroform comprises a mixture containing chloroform, methanol and deionized water. In some embodiments of the present application, the volume ratio of chloroform, methanol and deionized water is 1:1:1.

[0064] In a fifth aspect, the present application provides a method for multi-channel quantification of biomolecules. According to an embodiment of the present application, the method comprises: derivatizing the biomolecules by the method of the fourth aspect to obtain derivatized biomolecules; and analyzing the derivatized biomolecules to determine the content of the biomolecules. Thus, the method of the present application can directly realize six-channel parallel quantitative detection, greatly improve the throughput of complex sample analysis in the fields of proteomics, lipidomics, etc., and meet the demand for parallel processing of multiple samples. In addition, the tag is adjusted for isobaricity by the mass of the balancing group, combined with ion mobility separation and tandem mass spectrometry technology, can effectively distinguish isomers, reduce signal overlap interference, and accurately reflect the actual proportion of the target biomolecules.

[0065] In some embodiments of the present application, the analysis is performed by at least one of liquid chromatography, cyclic ion mobility separation, and multi-stage tandem mass spectrometry. In some embodiments of the present application, the analysis is performed by liquid chromatography, cyclic ion mobility separation, and multi-stage tandem mass spectrometry. Thus, the method of the present application combines liquid chromatography, cyclic ion mobility separation (cyclic IMS) and multi-stage tandem mass spectrometry (MS 2 CID + MS 3 CID), releases structure isomeric lipid head group characteristic fragment ions at the MS 2 CID stage, and realizes separation between isomers in the IMS dimension; further releases isomer tag channel-specific reporter ions at the MS 3 CID stage, to realize six-channel or even higher channel number quantitative analysis. This method overcomes the problem of insufficient structural resolution of traditional isobaric tags in lipid analysis, realizes accurate identification and quantification of lipid isomers without relying on ultra-high resolution instruments, and is suitable for high-throughput, low-cost, high-reproducibility lipidomics research. The present application provides a complete and efficient technical system for tag synthesis, lipid derivatization, and IMS-MS multi-dimensional separation and quantification, not only breaks through the bottleneck of the application of traditional isobaric tags in the field of lipids, but also greatly improves the throughput, sensitivity and accuracy of lipidomics analysis, and has a wide application prospect.

[0066] Embodiments of the present application are described in detail below. The embodiments described below are exemplary and are used to explain the present application, and cannot be understood as limiting the present application. If specific techniques or conditions are not mentioned in the embodiments, the techniques or conditions described in the literature in the art or according to the product manual are used. If the reagents or instruments are not mentioned by the manufacturer, they are all conventional products that can be obtained by purchase.

[0067] Instruments and parameters: The liquid chromatography was an ACQUITY UPLC I-Class PLUS system (Waters Corp., Wilmslow, U.K.) equipped with a degasser, two pumps, an autosampler, and a column oven. The lipids in the examples were separated by a reversed-phase chromatographic column, model: RPLC, CSH C18 column, 100 mm x 2.1 mm, 1.7 μm particle size (Waters Corp., Wilmslow, U.K.), with a column temperature of 50 °C. Mobile phase A: acetonitrile / water (60 / 40, by volume), mobile phase B: acetonitrile / isopropanol (10 / 90, by volume). Both mobile phases were supplemented with 10 mM ammonium acetate and 0.1% acetic acid. The mobile phase gradient was: 0-0.8 min: 50% B; 0.8-2 min: 50%~80% B; 2-2.3 min: 80% to 97% B; 3.2-8 min: 97% B; 8-8.5 min: 97% to 50% B; 8.5-11 min: 50% B. The flow rate of the mobile phase was 0.3 mL / min. The injection volume was 2 μL.

[0068] The mass spectrometer used was a SELECT SERIES cyclic IMS from Waters. The electrospray ionization-mass spectrometry settings were as follows: electrospray voltage +3500 V; cone hole voltage 40 V; interface heating temperature 250 °C; Trap CID energy 45 eV; Transfer CID 20 eV. The post-trap gradient was 5 V, the post-trap bias was 15 V, the helium cell entrance voltage was 3 V, the helium cell bias was 22 V, the array pulse height in eject was 15 V, and the pretransfer gradient was 5 V. During the cyclic ion mobility separation, the wave pulse height was set to 12 V, and the wave velocity was 375 m / s. The cIMS number of turns varied from 3 to 4. The following were not described in detail, but used the above mass spectrometry mode.

[0069] In phosphatidylethanolamine (PE 16:0_18:1) and phosphatidylserine (PS 16:0_18:1), "16:0" means a saturated fatty acyl chain with 16 carbon atoms and 0 double bonds, i.e., palmitic acid (C16:0). "18:1" means an unsaturated fatty acyl chain with 18 carbon atoms and 1 double bond, usually referring to oleic acid (C18:1Δ9). The underscore "_" separates the fatty acyl chains at the sn-1 and sn-2 positions (sn-1 or sn-2 is a unified numbering rule for carbon atoms on the glycerol backbone, with the left side defaulting to sn-1 and the right side to sn-2).

[0070] Example 1: Preparation of a six-channel tag The six-channel label backbone materials include 3-amino-4,4-dimethylvaleric acid, 2-amino-4,4-dimethylvaleric acid and 2-aminoheptanoic acid.

[0071] 1. Preparation of d4 channel tags: Three raw materials were dissolved separately in deionized water, followed by the addition of 2.5 equivalents of sodium cyanoborohydride. The mixture was cooled in an ice-water bath. As the solution temperature decreased, 2.5 equivalents of deuterated formaldehyde (20% by mass) were added dropwise, and the mixture was stirred in an ice-water bath for 45 minutes until the solution became clear and transparent. The reaction solution was then lyophilized to obtain the lyophilized product. The lyophilized product was dissolved in dichloromethane and purified by rapid column chromatography (mobile phase: methanol / dichloromethane). The methanol content in the mobile phase varied from 10% to 20% to 30% to 40% to 50%, with an elution volume of 10 mL for each ratio. The elution solution was dried under vacuum to obtain d4 channel tags containing three isomers, as shown below. Figure 1 As shown in the figure on the right.

[0072] 2. Preparation of d2 channel tags: The three raw materials are first processed separately... 18 O-exchange labeling is then used for subsequent reactions. Taking 100 mg of raw material as an example, the raw material is dissolved in 3 mL of H2 containing trifluoroacetic acid (8%, by volume). 18 In O, the solution was stirred at 65 °C for 20 hours. After the reaction was complete, the solution was dried under a nitrogen stream to remove most of the trifluoroacetic acid. The pH of the system was then adjusted to approximately 6 using 10 mM ammonium bicarbonate solution. Finally, the product was precipitated using cold diethyl ether. After filtration, the precipitated solid was repeatedly washed with cold diethyl ether until the product was pure, yielding the product... 18 Three skeleton materials marked with O.

[0073] Will be 18O-labeled three skeleton raw materials were dissolved in deionized water, then 2.5 equivalent cyanoborodeuteride sodium was added, and the mixture solution was cooled in an ice water bath. After the solution temperature was reduced, 2.5 equivalent formaldehyde (mass fraction 37%) was added dropwise, and the solution was stirred in an ice water bath for 45 minutes until it was clear and transparent. The reaction solution was dried by freeze-drying to obtain a freeze-dried product, which was dissolved in dichloromethane and purified by flash column chromatography (mobile phase: methanol / dichloromethane), with the methanol proportion in the mobile phase being 10%-20%-30%-40%-50%, and the elution volume of the mobile phase with different proportions being 10 mL. The elution solution was dried in a vacuum to obtain a d2 channel tag containing three isomers, as shown in Figure 1 as shown in the left graph.

[0074] By using steps 1 and 2 described above, a total of six tags were obtained. When used, the six tags were mixed with the labeled analysis sample, and six samples could be qualitatively and quantitatively detected in parallel.

[0075] In order to identify the structures of the six tags described above, nuclear magnetic detection was performed on the six tags, and the experimental results are shown in FIG. 2, wherein Figure 2a is a 2-(dimethylamino)heptanoic acid skeleton reporter ion m / z is a 130.123 channel tag; Figure 2b is a 2-(dimethylamino)heptanoic acid skeleton reporter ion m / z is a 132.143 channel tag; Figure 2c is a 2-(dimethylamino)-4,4-dimethylpentanoic acid skeleton reporter ion m / z is a 130.123 channel tag; Figure 2d is a 2-(dimethylamino)-4,4-dimethylpentanoic acid skeleton reporter ion m / z is a 132.143 channel tag; Figure 2e is a 3-(dimethylamino)-4,4-dimethylpentanoic acid skeleton reporter ion m / z is a 116.110 channel tag; Figure 2f is a 3-(dimethylamino)-4,4-dimethylpentanoic acid skeleton reporter ion m / z is a 118.123 channel tag. The results show that the three-channel isomer tag and the two-channel isomer tag are successfully coupled by the method of the present application.

[0076] Example 2: Derivatization of lipid standard Preparation of analysis sample: N,N-dimethylformamide solution containing 14 mM 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride, 6 mM 1-hydroxybenzotriazole and 14 mM N-methylmorpholine was prepared. 1 mg of single tag prepared in Example 1, Step 1 (for example, 2-amino-4,4-dimethylpentanoic acid as the backbone, lipid derivatization process as shown in Figure 3 a) was prepared. 191 μL of activation solution was prepared according to the molar ratio of tag, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole, N-methylmorpholine of 1:0.7:0.3:0.7, and incubated at room temperature for 30 minutes. The aminophospholipid was dissolved in anhydrous N,N-dimethylformamide containing 30% triethylammonium bicarbonate (TEAB) buffer in advance. After the tag activation was completed, the aminophospholipid was mixed with the activated tag reagent at a molar ratio of 1:800, and the reaction was continued at 60°C for 1 hour. After the aminophospholipid reaction was completed, 1.5 mL of a mixed solution of chloroform / methanol / deionized water (volume ratio 1:1:1) was added. The resulting mixture was vortexed for 10 minutes, and then centrifuged at 10,000 x g at 4°C for 10 minutes. The chloroform layer at the bottom was collected, dried and redissolved in methanol. The derivatized aminophospholipid reaction solution was sent to liquid chromatography-mass spectrometry analysis. Mass spectrometry analysis: The same concentration of aminophospholipid standard solution and the derivatized aminophospholipid reaction solution obtained in the above step were sent to reverse phase chromatography and connected to mass spectrometry for primary mass spectrometry detection in positive ion mode, with an injection volume of 3 μL. By comparing the mass spectrometry peak signals and chromatography peak areas of the lipid standards (PE 16:0 / 18:1 and PS 16:0 / 18:1) before and after derivatization, the derivatization efficiency and ionization efficiency improvement were evaluated.

[0077] Analysis results: The chromatograms of phosphatidylethanolamine PE 16:0_18:1 and phosphatidylserine PS 16:0_18:1 before and after derivatization are shown in Figures Figure 3 b, which shows that after derivatization, the lipid signal is improved by 2-3 times. The mass spectrometry results are shown in Figure Figure 4 b, which shows that after derivatization, the lipid signal is improved by 2-3 times. The mass spectrometry results are shown in Figure

[0078] Example 3: Feasibility verification of six-channel quantification Preparation of analysis sample: according to the procedure of Example 2, the six-channel tag prepared in Example 1 was used to derivatize phosphatidylethanolamine PE 16:0_18:1 respectively, and the derivatized lipids containing reporter ions were respectively configured m / z 116 / 118 channel (i.e. tag with 3-amino-4,4-dimethylpentanoic acid as the skeleton) at a concentration of 1:1 of derivatized lipids; containing reporter ions m / z 130 / 132 channel (i.e. tag with 2-amino-4,4-dimethylpentanoic acid as the skeleton) at a concentration of 1:2 of derivatized lipids; containing reporter ions m / z 130 / 132 channel (i.e. tag with 2-amino-4,4-dimethylpentanoic acid as the skeleton) at a concentration of 1:2 of derivatized lipids; containing reporter ions

[0079] Mass spectrometric analysis: the derivatized lipid mixture solution was sent to reverse chromatography and connected to mass spectrometry for tandem mass spectrometry-ion mobility detection in positive ion mode, with an injection amount of 3 μL.

[0080] Using the multi-dimensional fragmentation function of the SELECT SERIES cyclic IMS system, CID was first performed before mobility (Trap CID). Subsequently, the lipid head group fragments with isomer tag were subjected to 6-cycle circular mobility separation (IMS 6 , R~200) using the ring ion mobility technology, and the MS 2 CID spectrum mainly presented two characteristic fragmentation pathways: the isomer-labeled lipid head group directly dissociated to generate a lipid head group fragment with a tag label, and the carbon chain skeleton fragment generated by neutral loss of the lipid head group. After 6-cycle circular mobility separation by the SELECT SERIES cyclic IMS system, three characteristic mobility peaks were generated, corresponding to different isomers. After mobility separation, the isomer tag was further subjected to CID after mobility (Transfer CID). Using the unique time alignment parallel fragmentation function of the SELECT SERIES cyclic IMS system, pseudo MS 3 spectra of the mobility separated ions were synchronously obtained, and reporter ions indicating the content of the lipids were obtained for subsequent quantitative analysis.

[0081] Analysis results: the chromatograms before and after derivatization of phosphatidylethanolamine PE 16:0_18:1 with 2-amino-4,4-dimethylpentanoic acid as the skeleton are shown in Figure 5 a, and the results show that after derivatization, the lipid signal is improved by 2 times; the mass spectrum of derivatized phosphatidylethanolamine PE 16:0_18:1 by CID secondary fragmentation is shown in Figure 5As shown in b, the results indicate that the derivatized lipids exhibit a unique fragmentation pattern, namely, the generation of lipid head-base fragments with tags. The extracted ion mobility maps of the fragments from the three isomer-tagged derivatized products after cyclic IMS mobility separation are shown in Figure b. Figure 5 As shown in c, the results indicate that after six-cycle mobility separation, baseline separation can be achieved for different isomer tags; utilizing the time-aligned parallel fragmentation function unique to cyclic IMS, for m / z TAP CID fragmentation of the 297.1 fragment yielded tertiary mass spectra of different isomer tags, as shown below. Figure 5 As shown in d, the results show that the tagged lipid head base can be fragmented to obtain diagnostic ions.

[0082] Secondary mass spectrometry fragmentation results showed that the main fragment ions of the derivatized lipids were tagged lipid head groups. m / z 301.152. Ion mobility spectroscopy analysis as follows: Figure 6 The six-channel labeled lipid head fragments also produced three characteristic mobility peaks after six cycles of separation, corresponding to different isomers. Isotope labeling did not cause a significant mobility time shift (Δt < 0.1 ms). These three mobility peaks were analyzed by TAP MS. 3 CID. Each mobility peak generates a reporter ion: generated at 37.9 ms. m / z 116.110 / 118.123 (e.g.) Figure 5 (Mid-blue background mass spectrum); generated at 39.1 ms. m / z 130.123 / 132.143 (e.g.) Figure 5 (Mid-green background mass spectrum); generated at 40.6 ms. m / z 130.123 / 132.143 (e.g.) Figure 5 (Mass spectrum with orange background). By calculating the relative abundance of reporter ions within the same isomer channel, we obtained 1:1.07, 1:2.06, and 1:4.18, which are highly consistent with the initial mixing concentration ratios of 1:1, 1:2, and 1:4.

[0083] Example 4: Accuracy Verification of Six-Channel Quantitative Analysis Sample preparation: Following the procedure in Example 2, six parallel replicates of human normal colonic epithelial cell lipid extracts were derivatized using the six-channel tag prepared in Example 1. The extracted six-channel reaction solutions were mixed at a concentration ratio of 1:1:1:1:1:1, shaken well, and then sent to liquid chromatography-mass spectrometry (LC-MS). Simultaneously, the three underivatized parallel replicates of human normal colonic epithelial cell lipid extracts were diluted and directly sent to LC-MS.

[0084] Mass spectrometry analysis: The derivatized biological sample lipid solution was injected into reverse phase chromatography and connected to mass spectrometry for tandem mass spectrometry-ion mobility detection in positive ion mode, with an injection volume of 4 μL. The non-derivatized biological sample lipid solution was injected into reverse phase chromatography and connected to mass spectrometry for primary mass spectrometry detection in positive ion mode.

[0085] Using the multi-dimensional fragmentation function of the SELECT SERIES cyclic IMS system, CID was first performed before mobility (Trap CID). Subsequently, the lipid head group fragments with isomer tags were separated by 6-cycle circular mobility separation (IMS 6 , R~200) using the ring ion mobility technique, and the MS 2 spectra of the labeled lipids were obtained. The CID spectra mainly presented two characteristic fragmentation pathways: the isomer-labeled lipid head group directly dissociated to generate a lipid head group fragment with a tag label, and the carbon chain skeleton fragment generated by the neutral loss of the lipid head group. After 6-cycle circular mobility separation by the SELECT SERIES cyclic IMS system, three characteristic mobility peaks were generated, corresponding to different isomers. After mobility separation, the isomer tag was further subjected to CID after mobility (Transfer CID). Using the unique time alignment parallel fragmentation function of the SELECT SERIES cyclic IMS system, pseudo MS 3 spectra of the mobility-separated ions were synchronously obtained, and the reporter ions indicating the content of the lipids were obtained for subsequent quantitative analysis.

[0086] Analysis results: The results of lipid quantification using 6-channel tags are shown in Figure 7 The experimental reporter ion ratios were consistent with the expected ratios of 1:1:1:1:1:1, and the average RSD was less than 7%. Compared with the method of direct injection detection without derivatization (the average RSD was about 15%), the relative standard deviation was significantly reduced. Example 5: Establishment of quantitative curves for different amino phospholipids Preparation of analysis samples: According to the process of Example 2, the six-channel tags prepared in Example 1 were used to derivatize the lipid standards phosphatidylethanolamine PE 16:0_18:1 and phosphatidylserine PS 16:0_18:1, respectively. For the three isomer channels, each isomer channel was mixed according to the ratio of the two isobaric tags (reporter ion m / z 130.123 channel / reporter ion 132.143 channel) from 10%-30%-50%-70%-90% in increments, and after shaking, it was directly injected into liquid chromatography-mass spectrometry analysis.

[0087] Mass spectrometry analysis: The mixed solution was injected into reverse phase chromatography and connected to mass spectrometry for tandem mass spectrometry-ion mobility detection in positive ion mode, with an injection volume of 4 μL.

[0088] Using the multi-dimensional fragmentation function of the SELECT SERIES cyclic IMS system, CID was first performed before the drift (Trap CID). Subsequently, the lipid head group fragments with isomer tags were subjected to 6 cycles of cyclic drift separation (IMS 6 , R~200) using the ring ion drift technique, and the MS 2 spectra of the lipid markers were obtained. The CID spectra mainly presented two characteristic fragmentation pathways: isomer-labeled lipid head groups directly dissociated to generate lipid head group fragments with tag markers, and carbon chain skeleton fragments generated by neutral loss of lipid head groups. After 6 cycles of cyclic drift separation by the SELECT SERIES cyclic IMS system, three characteristic drift peaks were generated, corresponding to different isomers. After drift separation, the isomer tags were further subjected to CID after the drift (Transfer CID). Using the unique time alignment parallel fragmentation function of the SELECT SERIES cyclic IMS system, pseudo MS 3 spectra of the drift separated ions were synchronously obtained, and the reporter ions indicating the content of the lipids were obtained for subsequent quantitative analysis.

[0089] Analysis results: The mass spectrometry results were fitted with a curve, and the results are shown in Figure 8 The six quantitative curves established using phosphatidylethanolamine PE16:0_18:1 and phosphatidylserine PS 16:0_18:1 as models have good linearity, with R 2 all greater than 0.99, and the linear slopes are all close to 1, indicating that the relative proportions measured in the experiment can reflect the theoretical proportions. In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0090] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A tag for multichannel biomolecule quantification, characterized in that, include: First channel label and second channel label; The raw materials for the first channel tag and the second channel tag are each selected from two or more amino acid derivatives, and the amino acid derivatives include two or more amino acids that are isomers of each other; the first channel tag and the second channel tag are isoquants.

2. The label according to claim 1, characterized in that, The first channel label and the second channel label are made of the same material; Optionally, the amino acid derivative includes 3-amino-4,4-dimethylvaleric acid, 2-amino-4,4-dimethylvaleric acid, and 2-aminoheptanoic acid.

3. The label according to claim 2, characterized in that, The first channel tag includes a first reporter group, a first balancing group, and a first reactive group; Optionally, the second channel label includes a second reporter group, a second balancing group, and a second reactive group; Optionally, the first reporter group is selected from... , , At least one of them; Optionally, the first balancing group is selected from =O; Optionally, the first reactive group is selected from -COOH; Optionally, the second reporter gene is selected from... , , At least one of them; Optionally, the second balancing group is selected from = 18 O; Optionally, the second reactive group is selected from -C 18 O 18 OH.

4. The label according to claim 3, characterized in that, The first channel label is selected from at least one of the following: 、 、 ； Optionally, the second channel label is selected from at least one of the following: 、 、 。 5. A method for preparing the label according to any one of claims 1-4, characterized in that, include: Some amino acid derivatives were isotopically labeled to obtain isotopically labeled amino acids; The amino acid derivative or the isotope-labeled amino acid is subjected to a reductive methylation reaction to obtain a first channel tag or a second channel tag.

6. The method according to claim 5, characterized in that, The isotope labeling is performed in the following manner: The amino acid derivative was mixed with an isotope solution containing trifluoroacetic acid to obtain a mixture. The mixture is subjected to a first reaction treatment to obtain a first reaction product; The trifluoroacetic acid in the first reaction product is removed to obtain the second reaction product; The second reaction product was subjected to pH adjustment, precipitation, and washing in sequence to obtain the isotope-labeled amino acid.

7. The method according to claim 5, characterized in that, The amino acid derivative includes at least one of 3-amino-4,4-dimethylvaleric acid, 2-amino-4,4-dimethylvaleric acid, and 2-aminoheptanoic acid. Optionally, the reductive methylation reaction is carried out in the presence of reactants and a reducing agent; Optionally, the molar ratio of the amino acid derivative or the isotopically labeled amino acid, the reactant and the reducing agent is 1:(2-3):(2-3); Optionally, the reducing agent is selected from sodium cyanoborohydride or sodium cyanoborodeuteride; Optionally, the reactants are selected from formaldehyde or deuterated formaldehyde; Optionally, the amino acid derivative is subjected to a reductive methylation reaction, wherein the reducing agent is selected from sodium cyanoborohydride and the reactant is selected from deuterated formaldehyde; Optionally, the isotope-labeled amino acid is subjected to a reductive methylation reaction, wherein the reducing agent is selected from sodium cyanoborodeuteride and the reactant is selected from formaldehyde.

8. The method according to claim 7, characterized in that, The reductive methylation reaction further includes: freeze-drying the reductive methylation product; and purifying the freeze-dried product to obtain the first channel label or the second channel label. Optionally, the purification process is performed using column chromatography; Optionally, the mobile phase of the column chromatography is a mixture of methanol and dichloroethane.

9. The method according to claim 6, characterized in that, The mass-to-volume ratio of the amino acid derivative to the isotope solution containing trifluoroacetic acid is 100 mg:(1-5) mL; Optionally, the volume ratio of trifluoroacetic acid in the isotopic solution containing trifluoroacetic acid is 5%-10%; Optionally, the first reaction treatment is carried out at 60°C-70°C; Optionally, the first reaction treatment time is 15h-25h; Optionally, the pH is adjusted to 5.5-6.5; Optionally, the precipitation is carried out using cold diethyl ether; Optionally, the washing is performed using cold ether.

10. A reagent kit, characterized in that, This includes the label as described in any one of claims 1-4 or the label prepared using the method described in any one of claims 5-9.

11. A method for derivatizing biomolecules, characterized in that, include: The biomolecule is derivatized using the label according to any one of claims 1-4, the label prepared by the method according to any one of claims 5-9, or the kit according to claim 10 to obtain the derivatized biomolecule.

12. The method according to claim 11, characterized in that, The method includes: The first channel label and / or the second channel label in the label, or the first channel label and / or the second channel label in the kit, are activated respectively to obtain a first activated label and a second activated label; The first activation tag and / or the second activation tag are respectively mixed with the biomolecule to obtain a first mixed product and / or a second mixed product; The first mixed product and / or the second mixed product are respectively subjected to a second reaction treatment to obtain a third reaction product and / or a fourth reaction product; The third reaction product and / or the fourth reaction product are respectively subjected to a second mixing treatment with a mixture containing chloroform to obtain the third mixed product and / or the fourth mixed product; Centrifuge the third mixed product and / or the fourth mixed product to collect the trichloromethane layer solution respectively; The chloroform layer solution was dried to obtain the derivatized biomolecule.

13. The method according to claim 12, characterized in that, The activation treatment is carried out under the action of a catalyst; Optionally, the catalyst comprises 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 1-hydroxybenzotriazole, or N-methylmorpholine; Optionally, the molar ratio of the first channel label or the second channel label, the 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, the 1-hydroxybenzotriazole, and the N-methylmorpholine is 1:(0.5-1):(0.1-0.5):(0.5-1); Optionally, before the biomolecules undergo the first mixing treatment, the process further includes: pre-dissolving the biomolecules in anhydrous N,N-dimethylformamide containing triethylammonium bicarbonate buffer; Optionally, the molar ratio of the biomolecule to the first channel tag or the second channel tag is 1:(500-1000); Optionally, the second reaction treatment is carried out at 55°C-65°C; Optionally, the second reaction treatment time is 0.5h-1.5h; Optionally, the mixture containing chloroform includes chloroform, methanol, and deionized water; Optionally, the volume ratio of the trichloromethane, methanol, and deionized water is 1:1:

1.

14. A method for multichannel quantification of biomolecules, characterized in that, include: The biomolecule is derivatized using the method described in any one of claims 11-13 to obtain a derivatized biomolecule; The derived biomolecules are analyzed to determine their content.

15. The method according to claim 14, characterized in that, The analysis was performed using at least one of liquid chromatography, cyclic ion mobility separation, and multistage tandem mass spectrometry.

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