CN117969642A - Accurate relative quantitative analysis method and application of plant N-sugar chain - Google Patents
Accurate relative quantitative analysis method and application of plant N-sugar chain Download PDFInfo
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- CN117969642A CN117969642A CN202410394798.6A CN202410394798A CN117969642A CN 117969642 A CN117969642 A CN 117969642A CN 202410394798 A CN202410394798 A CN 202410394798A CN 117969642 A CN117969642 A CN 117969642A
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Abstract
The invention relates to the technical field of biological detection and analysis, and provides a method for accurately and relatively quantitatively analyzing N-sugar chains of plants and application thereof, wherein the method comprises the steps of (1) constructing a sample group for relatively quantitatively analyzing N-sugar chains of plants, and obtaining a detection map with mass spectrum signal intensity of N-sugar chain peaks of the plants reaching 10 4 and above; the mass displacement difference between the plant double-tag N-sugar chain internal standard and the plant N-sugar chain sample can reach 5Da or more; (2) The method comprises the steps of constructing a plant N-sugar chain sample and a plant double-label N-sugar chain internal standard relative quantitative analysis group, establishing a relative quantitative analysis formula of the content of each N-sugar chain based on the peak area of each N-sugar chain of the sample and the internal standard in a map, obtaining the content of the N-sugar chain with comparability, screening key N-sugar chains, and carrying out comparative analysis on the change of the content of all N-sugar chains on plant samples of different types, different tissue parts and different types and different batches of each growth and development stage.
Description
Technical Field
The invention relates to the technical field of biological detection and analysis, in particular to a method for accurately and relatively quantitatively analyzing N-sugar chains of plants and application thereof.
Background
Glycosylation is an important post-translational modification of proteins. The formation of glycoprotein （Strasser R, Seifert G, Doblin MS, Johnson KL, Ruprecht C, Pfrengle F, Bacic A, Estevez JM. Cracking the "Sugar Code": A Snapshot ofN- andO-Glycosylation Pathways and Functions in Plants Cells. Front Plant Sci. 2021 Feb 19;12:640919. doi: 10.3389/fpls.2021.640919. PMID: 33679857;）. by covalent binding of proteins with carbohydrates by the catalytic action of various Glycosyltransferases (GTs) and Glycosylhydrolases (GHs) the most common type of （Apweiler R, Hermjakob H, Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta. 1999 Dec 6;1473(1):4-8. ）, sugar chain in eukaryotic protein glycosylation modification is linked to asparagine (Asn) of the protein Asn-X-Ser/Thr sequence (Ser is serine, thr is threonine, X is any amino acid other than proline) is referred to as an N-sugar chain. N-sugar chains are classified into high mannose type, complex type, hybrid type and oligomannose type according to the composition and linking manner of the unused sugar residues （Lerouge P, Cabanes-Macheteau M, Rayon C, Fischette-Lainé AC, Gomord V, Faye L. N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol Biol. 1998 Sep;38(1-2):31-48.）.
N-glycomics is an emerging subject for systematically analyzing all N-sugar chains in a detection sample to obtain structural composition and relative quantitative information thereof, so as to characterize dynamic change law （Chau TH, Chernykh A, Ugonotti J, Parker BL, Kawahara R, Thaysen-Andersen M. Glycomics-Assisted Glycoproteomics Enables Deep and Unbiased N-Glycoproteome Profiling of Complex Biological Specimens. Methods Mol Biol. 2023;2628:235-263.）. of glycosylation of proteins in the life activity process, and along with the development of technology, a mass spectrum high-throughput detection technology gradually becomes a primary analysis tool for detection and analysis, wherein the mass spectrum high-throughput detection technology is to reflect the abundance ratio of sugar chains in the sample by comparing the relative intensity of mass spectrum peak signals after an internal standard is mixed with the sample to be detected during detection, so that the relative quantitative analysis of the N-sugar chains of the sample is performed. The relative quantitative analysis is a quantitative method for reflecting the change of the content of different samples to be detected relative to the multiple of the content of the same detection internal standard by taking the internal standard mixed in the sample as a reference and reflecting the relative change of the content of different samples according to the multiple relation different from the internal standard, so that the influence of errors caused by instrument detection and the like can be eliminated, the operation is simple and convenient, and the change condition of the content of N-sugar chains can be reflected. The relative quantitative analysis is divided into common relative quantitative analysis and accurate relative quantitative analysis, wherein the accurate relative quantitative analysis refers to that the mass displacement difference between an internal standard and a sample is more than 3Da, whereas the common relative quantitative analysis refers to that the mass displacement difference between the internal standard and the sample is less than or equal to 3Da.
In a quantitative analysis system of N-sugar chains of plants, various tissue parts such as leaves, roots, fruits, flowers and the like in the plants are rich in pigment substances, and various pigment substances such as anthocyanin, chlorophyll, carotenoid and the like in the plants participate in the regulation of plant color quality and volatile substance formation. The pigment has high ultraviolet light absorptivity and polarity, and the relative quantitative analysis accuracy ,（García-Gómez BE, Salazar JA, Nicolás-Almansa M, Razi M, Rubio M, Ruiz D, Martínez-Gómez P. Molecular Bases of Fruit Quality in PrunusSpecies: An Integrated Genomic, Transcriptomic, and Metabolic Review with a Breeding Perspective. Int J Mol Sci. 2020 Dec 30;22(1):333. doi: 10.3390/ijms22010333. PMID: 33396946; PMCID: PMC7794732.）, of the N-sugar chain is seriously affected during detection, so that the N-sugar chain of the plant body needs to be subjected to more complicated impurity removal and purification relative to a quantitative analysis system. Meanwhile, the cell wall is a characteristic structure of plant somatic cells, the components of the hard and thick support are mucilage complexes, the mucilage complexes exist outside cell membranes, the maintenance of cell morphology can enhance the mechanical strength of plants against external force, and in the process of extracting N-sugar chains, plant samples need more strict working procedures than animals to uniformly mix the samples. Therefore, extraction and purification of the N-sugar chain of the plant are more difficult than those of the animal body. In the aspect of analysis and detection, the detection and analysis mode currently used in animals is that the mass displacement difference between a metabolic marker and an enzymatic 18 O marker and a sample is less than or equal to 3Da, and the method belongs to common relative quantitative analysis and has the problems of poor accuracy, limited application objects, long cell culture period and complex operation. The metabolic markers and the enzymatic 18 O markers are applied to animals, the metabolic markers are applied to cell samples, specifically, an internal standard cell sample is placed in a culture medium for culture, amide- 15 N glutamine is added to the culture medium as a unique nitrogen source of the cell culture, the metabolic markers and the common amide- 14 N glutamine marked sugar chains can only form a 1Da mass displacement difference, a longer cell culture period is required, the medium is required to be replaced every day, only a 2Da mass displacement difference exists between the internal standard generated by the enzymatic 18 O markers and the sample to be detected, and when the internal standard is mixed with the sample to be detected, the isotope peak stack of 2-3 Da of the sample N-sugar chain to be detected is introduced into the culture medium, the accuracy of relative quantitative analysis is very poor (Cao W, Zhang W, Huang J, Jiang B, Zhang L, Yang P. Glycan reducing end dual isotopic labeling (GREDIL) for mass spectrometry-based quantitative N-glycomics. Chem Commun (Camb). 2015 Sep 14;51(71):13603-6. doi: 10.1039/c5cc05365j. PMID: 26240031.)., so that the limit of the state of the detection sample based on the metabolic markers and the small mass displacement difference of the enzymatic 18 O markers cause large interference in the analysis of the sample, and the accuracy of the relative quantitative analysis is poor.
At present, an accurate N-sugar chain extraction, enrichment and relative quantitative analysis system is designed based on sialic acid residue modification in animal bodies, but because the composition of a plant N-sugar chain is essentially different from that of animal bodies, the plant does not contain sialic acid residues, so that the accurate N-sugar chain relative quantitative method designed based on the composition of the animal N-sugar chain is not suitable for the plant N-sugar chain at all and cannot be used for reference by plants. It is therefore necessary to establish a precise isotope labeling quantitative system for plants. The composition of N-sugar chains of plants and animals is shown in FIG. 1, and the difference between them is specifically represented by the following 2 points: (1) N-acetylglucosamine of complex N-sugar chain of plant is modified by alpha-1, 3-fucose and beta-1, 2-xylose residues, and alpha-1, 6-fucose in mammal. (2) The terminal position of the N-sugar chain of the plant contains terminal Lewis-a epitopes, namely beta-1, 3-galactose and alpha-1, 4-fucose residues, which are not modified by sialic acid residues, whereas in mammals the terminal is usually modified by Lewis-x epitopes containing beta-1, 4-galactose and alpha-1, 3-fucose, and the terminal of the N-sugar chain of the mammal is modified by sialic acid residues. (He Jinxia, gu Xiaochen, wang Wenxia, et al) plant N-sugar chain detection technology research progress [ J ]. Biotechnology progress 2018,8 (6): 500-508. DOI:10.19586/j.2095-2341.2018.0119. The method based on sialic acid residue modification design is widely studied at present because the position, connection mode and number of sialic acid residue modification are related to characterization of various diseases such as Behcet disease and breast cancer, analysis of related N-sugar chains is of great significance to treatment studies of various diseases, and research from biological evolution finds that genes （Angata T, Varki A. Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem Rev. 2002 Feb;102(2):439-69.）. which are not involved in biosynthesis, activation or transfer of sialic acid residues in invertebrates are not suitable for plant bodies, the existing N-sugar chain relative quantitative analysis method based on sialic acid residue modification is not suitable for plant bodies, the N-sugar chain content in plant bodies is low and is easy to be interfered by impurities such as pigment of plant bodies, and the research on N-glycosylation in plant bodies is far behind animal bodies （Feng Y, Multiplex Quantitative Glycomics Enabled by Periodate Oxidation and Triplex Mass Defect Isobaric Multiplex Reagents for Carbonyl-Containing Compound Tags. Anal Chem. 2019 Sep 17;91(18):11932-11937;Seo N,. Isomer-Specific Monitoring of Sialylated N-Glycans Reveals Association of α2,3-Linked Sialic Acid Epitope With Behcet's Disease. Front Mol Biosci. 2021 Nov 23;8:778851．）.
At present, no accurate relative quantitative analysis method for N-sugar chains of plants exists. The existing analysis method of N-sugar chains of plants has no comparability among the same N-sugar chains of different batches and different samples, and cannot carry out systematic analysis on plant samples, for example, the analysis method cannot carry out systematic analysis on the glycosylation of proteins on samples of different plant types, different tissue parts and different growth and development stages of the same part, so as to regulate and control the physiological functions of the plants. The existing plant N-sugar chain relative quantitative analysis method is a non-standard quantitative method (Label free) for normalized representation of sugar chain abundance, and the sugar chain abundance refers to representing the content of different N-sugar chains in a sample by detecting and calculating the ratio of the peak area of a certain N-sugar chain in the sample to the peak area accumulated value of all N-sugar chains in the sample. The non-standard quantitative method is characterized in that the N-sugar chain content in an independent detection sample is not detected simultaneously with the sample group consisting of the N-sugar chain internal standard to carry out standard reference, so that the non-standard quantitative method can only analyze the relative abundance ratio （Choi, HY., Park, H., Hong, J.K. et al.N-glycan Remodeling Using Mannosidase Inhibitors to Increase High-mannose Glycans on Acid α-Glucosidase in Transgenic Rice Cell Cultures. Sci Rep 8, 16130 (2018); Wang T, Jia X, Liu L, Voglmeir J. 2021. Changes in protein N-glycosylation during the fruit development and ripening in melting-type peach. Food Materials Research 1: 2;）, between sugar chains in the samples detected in the same batch, but if different samples are detected and analyzed, such as samples of different plant types, samples of different tissue parts, sample detection of different growth stages of the same part and the like, due to unavoidable influencing factors such as instruments, detection environment, operation and the like existing in the detection of different batches, the content change of the same sugar chain in different samples is not comparable under the condition of no internal standard sugar chain reference, and the sugar chain contents of a plurality of samples cannot be compared and analyzed, so that the research of protein glycosylation on the regulation and control effect of the physiological functions of plants is severely limited. Another relatively accurate method for quantitative analysis of N-sugar chains of plants is a 15 N isotope quantitative metabolic labeling method, which has the problems of poor accuracy, limited application objects, long cell culture period and complex operation, and cannot be widely applied (Orlando R, Lim JM, Atwood JA 3rd, Angel PM, Fang M, Aoki K, Alvarez-Manilla G, Moremen KW, York WS, Tiemeyer M, Pierce M, Dalton S, Wells L. IDAWG: Metabolic incorporation of stable isotope labels for quantitative glycomics of cultured cells. J Proteome Res. 2009 Aug;8(8):3816-23.).
In summary, the extraction and purification of plant samples are more complex than that of animal bodies, and the detection and analysis modes are behind, so that no accurate method for relatively quantitatively analyzing plant N-sugar chains exists at present, and the problems of poor accuracy, limited application objects, long cell culture period and complex operation exist in the detection and analysis modes of metabolic markers and enzymatic 18 O markers in the prior art; the existing relatively accurate quantitative analysis method of N-sugar chains is designed based on sialic acid composition of animal N-sugar chains, and the plant does not contain sialic acid, so that the method is not suitable for N-sugar chains of plants at all and the plants cannot be referred to; the currently used plant N-sugar chain non-calibration quantitative analysis method has the problems that the culture period is long, the content change analysis of the same sugar chain in different samples cannot be performed, and the research of protein glycosylation on the regulation and control effect of plant physiological functions is severely limited.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a method for accurately and relatively quantitatively analyzing N-sugar chains of plants and application thereof, which constructs a sample group for effectively separating, purifying and extracting even N-sugar chains of plants, introduces-specific N-sugar chain samples for relatively quantitatively analyzing N-sugar chains of plants and relative quantitative analysis groups of N-sugar chain internal standards (Isotope INTERNAL STANDARD) of plants, ensures that plant samples can be fully and uniformly mixed, extracts the N-sugar chains which are not interfered by impurities such as plant pigments and the like, obtains high-efficiency purified and enriched N-sugar chains, simultaneously constructs double-tag internal standard detection in detection analysis, obtains N-sugar chain peaks of each sample in a detection map and corresponding internal standard peaks thereof to be paired, realizes accurate quantification of the N-sugar chain content of each sample by a specific relative quantitative analysis formula, can reflect the relative content of each N-sugar chain in different samples, can relatively quantitatively analyze the N-sugar chains of different plants, can be applied to different plant types, different plant growth stages and different sugar chain development stages, can be applied to different sugar chain development and basic analysis systems of different sugar chains and different types can be applied to a plurality of plant development and different enzyme-related analysis systems, and change physiological analysis systems can be used for the analysis and change of sugar chain development factors and the sugar chain analysis and the environmental analysis and have different effects, is important for further elucidating the regulating and controlling functions of the physiological functions of the plants by glycosylation of the proteins.
The invention provides a method for accurately and relatively quantitatively analyzing N-sugar chains of plants and application thereof, comprising the following steps:
(1) Construction of sample groups for relative quantitative analysis of N-sugar chains of plants: the sample group comprises a plant N-sugar chain sample and a plant double-tag N-sugar chain internal standard, wherein the plant N-sugar chain sample to be detected is subjected to the steps of separation, enzyme digestion, N-sugar chain release and purification to obtain a plant N-sugar chain sample, so as to obtain a detection map with the mass spectrum signal intensity of a plant N-sugar chain peak reaching 10 4 and above; separating, enzyme cutting, releasing N-sugar chains, double-tag marking reaction and purifying the crude sample of the plant N-sugar chains to be detected to obtain plant double-tag N-sugar chain internal standards, wherein the mass displacement difference between the plant double-tag N-sugar chain internal standards and the plant N-sugar chain sample can reach 5Da or more;
(2) Construction of plant N-sugar chain samples and plant ditag N-sugar chain internal standard relative quantitative analysis groups: and mixing and detecting the plant N-sugar chain sample and the plant double-tag N-sugar chain internal standard to obtain a mass spectrum map, and establishing a ratio relation between each N-sugar chain in different samples and the peak area of the corresponding internal standard based on the peak area of each N-sugar chain in the plant N-sugar chain sample and the corresponding plant double-tag N-sugar chain internal standard in the map, so as to reflect the relative content of the same N-sugar chain in different samples.
In one embodiment of the present invention, the formula for the relative quantitative analysis of the N-sugar chain content is ：A1=Na1/Na1 Internal standard ,A2=Na2/Na2 Internal standard ,……An= Nan/Nan Internal standard ……;
Wherein A 1、A2、……An is the relative content of 1,2, … … N N-sugar chain peaks in the sample; na 1、Na2、……Nan is the peak area of 1,2, … … N N-sugar chains in the sample, and Na 1 Internal standard 、Na2 Internal standard 、……Nan Internal standard is the peak area of 1,2, … … N N-sugar chains in the N-sugar chain internal standard; na 1 and Na 1 Internal standard are N-sugar chain peaks appearing in pairs with a mass shift difference of 5Da in the spectrum, and among a pair of N-sugar chain peaks, na 1 is small in mass and Na 1 Internal standard is large in mass.
In one embodiment of the invention, the purification refers to the purification of enriched plant N-sugar chains using a C18, PGC solid phase extraction column.
In one specific embodiment of the present invention, the dual-tag labeling reaction refers to that the enzymatic isotope labeling is performed simultaneously with the release of the N-sugar chain, and the redox ring-opening reaction is performed after the release of the N-sugar chain is completed, so as to obtain the plant dual-tag N-sugar chain internal standard with a mass shift difference of 5 Da.
In one specific embodiment of the invention, the step of the double-tag labeling reaction is to dissolve the polypeptide after enzyme digestion in an acidic buffer solution of H 2 18 O, and add PNGase A to react the N-sugar chain end with 18 O tag; then NaBD 4 is added to enable the N-sugar chain end to generate oxidation-reduction ring-opening reaction, so as to obtain the plant double-label N-sugar chain internal standard with the mass shift of 5 Da.
In one specific embodiment of the invention, the separation method is to extract the total plant protein from the crude N-sugar chain sample of the plant to be detected by using a Buffer Y Buffer solution, and remove pigment impurities in the total plant protein by using methanol and acetone to obtain the plant protein.
In one specific embodiment of the invention, the method for enzyme digestion is to use trypsin and chymotrypsin to enzyme-digest plant proteins to obtain plant polypeptide samples.
In one embodiment of the invention, the method for releasing N-sugar chains is to use PNGase A reagent to enzyme-cut the obtained plant polypeptide sample to release plant N-sugar chains.
In one embodiment of the present invention, in step (2), the plant N-sugar chain sample and the solution of the plant ditag N-sugar chain internal standard are mixed in a volume ratio of 1:1.
In one embodiment of the present invention, in step (2), the mass spectrum comprises any one or more of matrix-assisted laser desorption ionization mass spectrometry, electrospray mass spectrometry, tandem mass spectrometry, and multi-stage mass spectrometry.
Further, the separation method specifically comprises the following steps:
The invention carries out liquid nitrogen freezing and grinding to obtain uniform powder, and stores at-80 ℃. Weighing 0.5-5 g of plant sample, adding 3-4 times of protein extract, and swirling. The composition of the protein extract is as follows: 1M Tris-HCl (ph=8.2), 10% (w/v) Sodium Dodecyl Sulfate (SDS), 1% (v/v) beta-mercaptoethanol, 0.5M Ethylene Glycol Tetraacetic Acid (EGTA), 0.5M Ethylene Diamine Tetraacetic Acid (EDTA), 0.1M phenylmethanesulfonyl fluoride (PMSF), and 1x protease inhibitor cocktail. The mixed solution after vortex is heated for 20mins in a water bath at 65 ℃, the supernatant is centrifugally taken and transferred to a new test tube, then an equal amount of precooled phenol buffer solution (PH=7.5-7.9) is added, after vortex is uniform, the mixture is centrifugally treated for 15mins at 12,000 rpm, and the upper phase water phase is removed. The lower organic phase was again washed twice with 50mM Tris-HCl (ph=7-9) buffer and the procedure was as above. The phenol phase was then mixed with 5 volumes of pre-chilled 0.1M ammonium acetate MeOH solution and the protein was precipitated by standing overnight at-20 ℃. Washing the obtained protein precipitate with precooled MeOH and acetone twice in sequence, and freeze-drying to obtain total protein, and preserving at-80 ℃;
Further, the method for enzyme digestion specifically comprises the following steps:
The total protein obtained was dissolved in 3-4 ml of 0.01M Tris-HCl buffer (pH=8) and the protein was inactivated by heating at 100℃for 5 min. The protease was cleaved into polypeptides by addition of 0.01M Tris-HCl configurations 30 mg/ML TRYPSIN, 30 mg/ml chymotrypsin and 0.2M CaCl 2 at 37℃for reaction 16 h. After the reaction, the enzyme is deactivated by heating at 100-120 deg.C for 5 min. 5000 Centrifuging at normal temperature for 5min, taking supernatant, detecting the concentration of the polypeptide by using Nanodrop, quantifying the content of the polypeptide by 1-3 mg, and rotationally evaporating the quantified polypeptide to dryness at the maximum of 30-45 ℃.
Further, the method for labeling reaction of the double labels specifically comprises the following steps:
The evaporated polypeptide is redissolved in an acidic buffer solution with the solvent of H 2 18 O of 0.1-1M PH =4.0, and 2.5-5U/G FW PNGASE A is added to react at 37 ℃ for 24H, so that the N-sugar chain end cleaved from the N-glycopeptide is reacted with 18 O label. After the reaction, the enzyme was deactivated by heating at 100℃to 5 min. Adding NaBD 4 of 0.2-0.3 mmol, reacting at 65 ℃ for 2 hours, and carrying out redox ring-opening reaction on the tail end of the N-sugar chain. The N-sugar chain tag adds a total of 5Da mass shift difference compared to the untagged N-sugar chain.
Further, the purification method specifically comprises the following steps: purifying N-sugar chains by using a C18 solid phase extraction column, enriching N-sugar chains by using a PGC column, and re-dissolving in 20-25 mu l H 2 O after vacuum spin-steaming.
The invention also provides an application of the method in N-glycomics of plants, which comprises the following steps:
(1) Respectively obtaining relative quantitative data of N-sugar chains from different samples by using the accurate relative quantitative analysis method of the N-sugar chains of the plants;
(2) Comparing and analyzing relative quantitative data of the same N-sugar chains in all samples, wherein the same N-sugar chains refer to N-sugar chains with the same mass displacement and error within +/-0.4 in a detection map of all samples, so as to meet the significance difference (SIGNIFICANT DIFFERENCE) p <0.05, screening the difference N-sugar chains, and analyzing the effect of the difference N-sugar chains on the regulation and control of plant physiological processes. The significance difference is an evaluation of the data difference and can be calculated by a t-test (Student t-test) method.
Furthermore, the invention also provides an application of the method in fruit ripening research; preferably, the fruit is tomato;
Further, the specific application steps in tomato fruit ripening research are as follows:
1) Three biological repeats are respectively arranged on tomato fruits in the development stages of a green ripening stage (IMG), a color breaking stage (BR) and a red ripening stage (BR 15) after 15 days of color breaking, 9 tomato fruits which have no mechanical injury and uniform plant diseases and insect pests are taken from each biological repeat, N-sugar chains are extracted, and the tomato fruits in the color breaking stage are prepared and detected as internal standards;
2) Mixing tomato fruit sample to be detected with internal standard sample in equal proportion, and using N-sugar chain structure analysis and identification software Glycomod to identify a pair of sugar chain peaks with equal mass displacement difference, wherein the small mass is N-sugar chain to be detected, and the other one generates 5Da mass as quantitative internal standard peak of the sugar chain. After obtaining relative quantitative values, comparing and analyzing the relative quantitative values of the same N-sugar chains in all detection samples to meet the p <0.05 screening difference N-sugar chains;
further, in the step 2), the method specifically includes:
The N-sugar chain detection matrix was prepared by using 0.01M of 2, 5-dihydroxybenzoic acid in methanol, and the detection instrument was a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (UlrafleXtremeTM MALDI-TOF/TOF MS) equipped with Smartbeam Nd:YAG (355 nm) laser. The target plate adopts 384 polishing plates (MTP 384 polished steel), mass spectrum data analysis adopts Bruker Flexanalysis 3.4.4, and 18N-sugar chains are identified by identifying the change of maturation-related specific polysaccharide in3 tomato development stage samples, and 7 marked N-sugar chains (H2N 2F1X1, H3N2F0X1, H3N2F1X0, H3N2F1X1, H4N2F0X1, H7N7F0X0 and H3N4F1X 1) with obviously up-regulated abundance are screened out by taking p <0.05 as a screening condition in the development and maturation process of tomato fruits, so that the N-glycosylation analysis plays a very important role in the regulation and control of plant physiological processes.
The beneficial effects of the invention are as follows:
(1) The method for relatively quantitatively analyzing the N-sugar chains of the plants constructs a specific sample group for relatively quantitatively analyzing the N-sugar chains of the plants, a plant N-sugar chain sample and a plant double-label N-sugar chain internal standard relatively quantitatively analyzing group, so that each N-sugar chain peak of the samples in the detection map and the corresponding internal standard peak are in pairs, the N-sugar chain content with comparability is obtained through a specific relatively quantitative analysis formula, the plant N-sugar chain content of each sample is accurately quantified, the relative content of each N-sugar chain in different samples can be reflected, the comparative analysis of the content change of all N-sugar chains can be carried out on different plant samples of different types, different tissue parts and different batches in different growth stages, the content change analysis of all N-sugar chains is not influenced by unavoidable error factors such as instruments, detection environments, operations and the like, the method can be applied to the screening of key N-sugar chains of the plants, the physiological processes of various sugar residues in the plant growth and the like can be applied to elucidate the regulation and control functions of various sugar residues of sugar residues in the plant, and the physiological functions are very important to the regulation and control of sugar enzyme processing.
(2) According to the method, the N-sugar chains in the tomato ripening process are relatively quantitatively analyzed, and the key N-sugar chains related to fruit ripening are screened out, so that the quality of the fruits is improved, the postharvest shelf life is prolonged, and the economic loss is reduced.
Drawings
FIG. 1 is a diagram showing an example of complex N-sugar chain structures of plants and mammals.
FIG. 2 is a diagram showing a method of analyzing the relative amounts of N-sugar chains of plants.
FIG. 3 is a graph showing the results of detection of the reaction conditions for relative quantitative analysis of N-sugar chains in plants; wherein A in the figure 3 is a MALDI-TOF mass spectrum of the maltohexaose at different reaction temperatures and times with different amounts of NaBH 4; b in FIG. 3 is a MALDI-TOF mass spectrum of horseradish peroxidase with a shift difference of 2Da of double-label 5Da of enzymatic 18 O label, 3 Da NaBD 4 redox label and enzymatic 18 O label combined with NaBD 4 redox.
FIG. 4 is a graph of the relative quantitative accuracy validation of the horseradish peroxidase isotope labeling method; wherein a in fig. 4 is a MALDI mass spectrum of horseradish peroxide high-abundance N-sugar chain H3N2F0X 1; b in FIG. 4 is a bipartite plot of the theoretical and corresponding measured ratios of two N-sugar chains (H3N 2F0X1, H3N2F1X 1) of horseradish peroxidase.
FIG. 5 is a graph showing the relative quantitative results of the application of the double-labeled internal standard to N-sugar chains in three developmental stages of tomato IMG, BR, BR; wherein, A in FIG. 5 is a relative quantitative MALDI-TOF mass spectrum of tomato IMG, BR, BR at three developmental stages; b in FIG. 5 is a bar graph of the relative amounts of sugar chains of tomato IMG, BR, BR at three developmental stages.
Detailed Description
Samples, reagents and the like used in the examples described below are commercially available unless otherwise specified. The method can effectively avoid impurity interference of plant pigment and the like by separating, is suitable for obtaining oligomannose, high mannose, heterozygote and complex plant N-sugar chains from all plant polypeptides by enzyme digestion, purifying and enriching the oligomannose, high mannose, heterozygote and complex plant N-sugar chains, obtaining a specific internal standard and a plant sample construction sample group by double-tag marking, obtaining a detection map meeting the requirements, analyzing the content of the N-sugar chains by utilizing a formula based on the relative quantitative analysis group constructed by the sample and the internal standard, and further comparing the content of the same N-sugar chains among different samples, screening key N-sugar chains, researching plant physiological functions, and ensuring that the plant has genetic high conservation. Among them, tomato is one of the most widely planted commercial crops in the world, the annual output exceeds 1 hundred million and 8 million tons, and the genetic resource is rich and the growth period is short, so it is commonly used in research of fruit development, secondary product metabolism, and plant-microorganism interaction. The tomato Fruit genome is simple diploid, has high genetic conservation, has a short planting and culturing period (J.J. Giovannoni, Genetic regulation of fruit development and ripening, Plant Cell 16(2004) S170–S180.),, is one of common horticultural crops, is a main source of essential nutrients of human diet, and has extremely high economic value (Yudong Liu, the Molecular Regulation of ETHYLENE IN circuit ring, 2020). The fruit ripening process is one of the key stages for improving the quality indexes such as nutrition, flavor and the like of the fruits. The current research on fruit ripening mainly focuses on the expression of various processing enzymes in N-glycosylation processes of （Li S, Chen K, Grierson D. Molecular and Hormonal Mechanisms Regulating Fleshy Fruit Ripening. Cells. 2021 May 8;10(5):1136.）. plants such as hormone, histone marks and DNA methylation, which can promote the fruit ripening, and the research on N-glycosylation modification of proteins as important post-translational modification pathways of organisms in fruit ripening is limited.
The plants in the specific embodiments of the present invention will be described with reference to the application of the present invention in the ripening of tomato fruits, but the present invention is not limited thereto and the present invention is applicable to all plants.
Acetone, ammonium acetate, ethylenediamine tetraacetic acid (EDTA), ethylene Glycol Tetraacetic Acid (EGTA), heavy oxygen water (H 2 18 O) were purchased from ala Ding Gongsi (Shanghai, china). Tris saturated phenol solution (ph=7.7-8.1) was purchased from biological engineering (Shanghai) limited (Shanghai, china). 1M Tris-HCl buffer (pH=8.2) was purchased from Fried company (Hangzhou, china). Porous graphitized carbon ENVI-Carb solid phase extraction column (PGC, 250 mg) was purchased from the chromatographic company (Bellefonte, USA). C18 solid phase extraction cartridge (500 mg) was purchased from Waters company (Massachuset, USA). PNGase A (500U/μl) was purchased from New England Biolabs (Ipswich, mass., USA). NaBD 4, protease inhibitors, trypsin, HPLC grade methanol (MeOH), acetonitrile (ACN) were purchased from merck corporation (Darmstadt, germany). Purified deionized water uses the Milli-Q system (Milford, mass., USA). Horseradish peroxidase was purchased from source leaf company (Shanghai, china).
The following abbreviations or foreign language terms are used throughout this invention:
IMG, 20 days after flowers;
BR, fruit color breaking period;
BR15, 15 days after the fruit breaking period;
CV, coefficient of variation;
ddH 2 O, deionized water;
DHB, 2, 5-dihydroxybenzoic acid;
HRP, horseradish peroxidase;
MeOH, methanol;
MS, mass spectrometry;
m/z, mass to charge ratio;
PNGase a, peptide N-glycosidase a;
R 2, correlation coefficient;
rpm, revolutions per minute;
Tris-HCl, tris (hydroxymethyl) aminomethane hydrochloride;
mu l, microliters;
M means mol/L
Example 1: plant N-sugar chain double-label marking method
As shown in FIG. 2, the 5Da double-label internal standard increases the mass displacement difference of the internal standard peak, avoids the superposition of the internal standard peak and the 3Da label peak of the peak to be detected, and improves the accuracy of relative quantitative analysis of N-sugar chains in a sample. Enzymatic 18 O labeling is a mass spectrum-based sugar chain relative quantification means with a large application prospect in recent years. Compared with chemical markers and metabolic markers, the enzymatic 18 O markers occur in the process of releasing sugar chains by enzymes, and only a heavy oxygen water system is needed to be added into an enzyme digestion system, so that the operation is simple and efficient, side reactions are avoided, and the cost is saved. The internal standard of the enzymatic 18 O mark is easy to produce isotope stacking when mass spectrum is detected relatively quantitatively due to small mass displacement difference, so that the relative quantitative error is increased. To optimize detection of defects with enzymatic 18 O markers, the present invention employs a method that combines enzymatic 18 O markers with chemical redox markers. The reducing agent NaBD 4 with low cost and strong effect is selected to reduce the N-sugar chain reducing end hemiacetal into hydroxyl, and the mass increment of 2Da is introduced. Meanwhile, one covalent bond hydrogen atom at the reducing end of the N-sugar chain is replaced by one deuterium atom in the reduction reaction, resulting in an additional 1Da mass increment. Therefore, as shown in fig. 2, the 5Da dual-label internal calibration method of the invention increases the mass displacement difference between the peak to be detected and the internal standard peak, so that each N-sugar chain peak in the sample to be detected has an accurate internal standard peak with the same ionization efficiency.
The preparation method of the 5Da double-tag internal standard is combined with the step of polypeptide enzyme digestion of N-sugar chains in the process of extracting plant proteins, when the N-sugar chains are digested after the polypeptide is extracted, 18 O is reacted on the tail ends of the N-sugar chains of plants during the N-sugar chain digestion, and after the enzyme digestion is finished, naBD 4 is added for oxidizing and reducing the N-sugar chains at the tail ends of the plants. The loss generated by the reaction with NaBD 4 acid and alkali is reduced, the polypeptide redissolution solvent system is reduced, and the polypeptide is redissolved by a 1M citric acid buffer solution system (PH=4) with 50 mu l of heavy oxygen water as a solvent.
The invention explores the influence of factors such as reaction temperature, reaction time, reagent concentration and the like on the reaction efficiency of the reduction-end isotope. Pre-experiment the invention selects the maltose (G6) with excessive ratio to the actual sample as the standard substance to be reduced and opened with NaBH 4, and the reaction condition is searched. As shown in FIG. 3A, the present invention set 4 reaction conditions, 0.1 mmol NaBH 4 at room temperature for 18h,0.1 mmol NaBH 4 at 65℃for 2h, and 0.16 mmol, 0.2 mmol NaBH 4 at 65℃for 2 h. As shown in FIG. 3A, naBH 4 of 0.2 mmol was treated at 65℃for 2 hours, and a G6 signal predictive of 2Da mass-shift redox ring opening was detected after reduction, with a derivatization efficiency approaching 100%.
After the reaction conditions of the double-tag internal standard are determined, horseradish peroxidase is selected as a standard protein, and after enzyme digestion is performed on the polypeptide, the content of 1mg of the polypeptide equivalent to that of an actual sample is quantified. As shown in B in FIG. 3, the N-sugar chain peak with the molecular weight of 1211m/z in the main peak of the partially amplified HRP is taken as a reference, and the enzymatic 18 O marker, the NaBD 4 redox marker and the enzymatic 18 O marker combined with the NaBD 4 redox marker respectively generate 2Da, 3Da and 5Da internal standard peaks, and the MALDI-MS detection result shows that the N-sugar chain internal standard of the HRP is increased by 5Da molecular weight compared with an untreated sample after being subjected to 5Da isotope labeling treatment. The reaction condition of the 5Da double-label internal standard is basically established, and the method is suitable for various detection instruments and has wider application.
Example 2 verification of the accuracy of the plant double tag marking method
In order to verify the linearity and accuracy of an isotope labeling method of N-sugar chain 5Da molecular weight, plant N-glycoprotein standard substance horseradish peroxidase (HRP) is selected as a standard substance protein, isotope labeling is carried out on 5Da (H 2 18O+NaBD4) in a reaction, and the volume ratio (10:1, 5:1, 1:1, 1:5 and 1:10) of mixing 5 internal standards with a sample is set during mass spectrometry. Analyzing the N-sugar chain result detected by HRP, selecting two N-sugar chain peaks (H3N 2F0X1 and H3N2F1X 1) with stronger detection intensity for statistical analysis, calculating the isotope quantitative peak calculation ratio, and generating a bipartite graph between the theoretical ratio of the sample and the internal standard and the corresponding measurement ratio.
As shown in A in FIG. 4, after HRP standard and internal standard are mixed in different volume ratios, the molecular weight of the amplified map of 1211 sugar chain peak, and the quantitative peak detection intensity of the N-sugar chain peak with the molecular weight of 1211 added with 5Da molecular weight shows corresponding ratio change with different volume ratios. As shown in B in fig. 4, the results have good linear relationship in the range of two orders of magnitude from 1:10 to 10:1, and the correlation coefficients are all greater than 0.99. As shown in Table 1, the quantitative results were highly accurate, and the Y-axis intercept ranges of the two N-sugar chains were 0.03211 and 0.05263, respectively, and the Coefficient of Variation (CV) ranges were 7.16% and 4.25%, respectively. Therefore, the relative quantification of the double-label internal standard capable of generating the 5Da displacement difference can not cause error interference on N-sugar chains due to isotope peak stacking, and the accuracy of a quantification result is improved.
TABLE 1 quantitative determination results of horseradish peroxidase representative N-sugar chains and internal standards at different ratios
Example 3: preparation of plant high-flux double-tag internal standard
The invention prepares the double-tag internal standard of the detection sample of the tomato developmental stage by using the sample of the tomato Breaker stage, discusses the marked N-sugar chain of the tomato in the developmental stage process, and provides more accurate reference basis for the research of N-glycosylation in the field of plant developmental maturity.
1G of fresh sample was weighed out, and vortexed in 3ml of protein extract. BufferY extracting protein, heating the mixed solution after vortex in a water bath at 65 ℃ for 20min after separation, centrifuging to obtain supernatant, transferring the supernatant to a new test tube, adding an equal amount of precooled phenol buffer solution (PH=7.5-7.9), centrifuging at 12,000 rpm for 15min after vortex uniformity, and removing an upper phase water phase. The lower organic phase was again washed twice with 50mM Tris-HCl (ph=7-9) buffer and the procedure was as above. The phenol phase was then mixed with 5ml of 0.1M ammonium acetate MeOH solution and left overnight at-20℃to precipitate the BR stage of tomato protein. The total protein was dissolved in 0.01M Tris-HCl buffer (pH=8) and the protein was inactivated by heating at 100℃for 5 min. The protease was cleaved into polypeptides by addition of 0.01M Tris-HCl at 37℃for 16h in configurations 30. 30 mg/ML TRYPSIN, 30 mg/ml chymotrypsin and 0.2M CaCl 2. After the reaction, the enzyme was deactivated by heating at 100℃for 5 min. Centrifuging at 5000rmp at normal temperature for 5min, collecting supernatant, detecting the concentration of tomato polypeptide at BR stage by Nanodrop, quantifying 1mg polypeptide content, and rotary evaporating the quantified polypeptide at 45deg.C.
The evaporated polypeptide was redissolved in 1M citrate buffer (ph=4.0) with solvent H 2 18 O, and 0.5 μl PNGase a was added to react 24H at 37 ℃ to react the BR stage tomato sample N-sugar chain end with 18 O tag. After the reaction, the enzyme was deactivated by heating at 100℃for 5 min. 90 μl of NaBD 4 (2M) was added and reacted at 65℃for 2 hours, and the N-sugar chain end was subjected to a redox ring-opening reaction to increase the amount by 3Da. The N-sugar chain tag adds a total of 5Da mass shift difference compared to the untagged N-sugar chain. Purifying N-sugar chain by using a C18 solid phase extraction column, enriching N-sugar chain by using a PGC column, and freezing and preserving the tomato internal standard sample at-20 ℃ after redissolving at 20 mu l H 2 O after vacuum spin-steaming.
Example 4: relative quantitative analysis and comparison of N-sugar chains of tomato fruits at different stages
According to the invention, three development stage samples of tomatoes IMG, BR, BR are selected, N-sugar chains are extracted, and double-label internal standards are prepared in a tomato color breaking stage. As shown in FIG. 5A, each N-sugar chain peak has an internal standard peak corresponding to the same intensity, and the sugar chain compositions of the three stages have obvious differences. As shown in Table 2, 18 different N-sugar chains were identified in total, including information identifying the sugar unit composition, sugar structure prediction, and original molecular weight of the N-sugar chains. The N-sugar chain glycoforms mainly identified in the development stage of tomato fruits are complex N-sugar chains. And (3) carrying out relative quantitative analysis on the identified N-sugar chains and the corresponding N-sugar chain internal standard peaks, and comparing the variation difference of IMG, BR, BR corresponding to the sugar chains as shown in B in fig. 5, so that 7N-sugar chains in the development and maturation process of tomato fruits can be screened out to show obvious differences and all show up-regulation trends. It was found that 7N-sugar chains exhibited significant differences, including oligomannose type N-sugar chains (H2N 2F1X1, H3N2F0X1, H3N2F1X0, H3N2F1X 1), heterozygous type N-sugar chains (H4N 2F0X 1), high mannose type (H7N 7F0X 0), complex type N-sugar chains (H3N 4F1X 1) and as fruits develop mature, the 7N-sugar chain abundance increased significantly. In agreement with previous studies (Priem and Gross, 1992), oligosaccharide chains containing xylose modifications were able to promote tomato maturation results.
The invention screens out 7 important N-sugar chains in the process of tomato fruit development and maturation by a precise relative quantitative analysis method of N-sugar chains in plant samples by a double-tag internal standard with the mass displacement difference of up to 5Da and a constructed specific segmented homologous polysaccharide group, and provides support for researching N-glycosylation and fruit growth and development.
TABLE 2 quantitative determination of tomato developmental stage 18N-sugar chain information tables
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Claims (10)
1. The accurate relative quantitative analysis method of the plant N-sugar chain is characterized by comprising the following steps of:
(1) Construction of sample groups for relative quantitative analysis of N-sugar chains of plants: the sample group comprises a plant N-sugar chain sample and a plant double-tag N-sugar chain internal standard, wherein the plant N-sugar chain sample to be detected is subjected to the steps of separation, enzyme digestion, N-sugar chain release and purification to obtain a plant N-sugar chain sample, so as to obtain a detection map with the mass spectrum signal intensity of a plant N-sugar chain peak reaching 10 4 and above; separating, enzyme cutting, releasing N-sugar chains, double-tag marking reaction and purifying the crude sample of the plant N-sugar chains to be detected to obtain plant double-tag N-sugar chain internal standards, wherein the mass displacement difference between the plant double-tag N-sugar chain internal standards and the plant N-sugar chain sample can reach 5Da or more;
(2) Construction of plant N-sugar chain samples and plant ditag N-sugar chain internal standard relative quantitative analysis groups: and mixing and detecting the plant N-sugar chain sample and the plant double-tag N-sugar chain internal standard to obtain a mass spectrum map, and establishing a ratio relation between each N-sugar chain in different samples and the peak area of the corresponding internal standard based on the peak area of each N-sugar chain in the plant N-sugar chain sample and the corresponding plant double-tag N-sugar chain internal standard in the map, so as to reflect the relative content of the same N-sugar chain in different samples.
2. The method according to claim 1, wherein the formula for the relative quantitative analysis of the N-sugar chain content is ：A1=Na1/Na1 Internal standard ,A2=Na2/Na2 Internal standard ,……An= Nan/Nan Internal standard ……;
Wherein A 1、A2、……An is the relative content of 1,2, … … N N-sugar chain peaks in the sample; na 1、Na2 、……Nan is the peak area of 1,2, … … N N-sugar chains in the sample, and Na 1 Internal standard 、Na2 Internal standard 、……Nan Internal standard is the peak area of 1,2, … … N N-sugar chains in the N-sugar chain internal standard; na 1 and Na 1 Internal standard are N-sugar chain peaks appearing in pairs with a mass shift difference of 5Da in the spectrum, and among a pair of N-sugar chain peaks, na 1 is small in mass and Na 1 Internal standard is large in mass.
3. The method according to claim 1, wherein the double-label labeling reaction is to perform enzymatic isotope labeling simultaneously with the release of the N-sugar chain, and then perform redox ring opening reaction after the release of the N-sugar chain is completed, so as to obtain a plant double-label N-sugar chain internal standard with a mass shift difference of 5 Da.
4. The method according to claim 1, wherein the separation method is to extract total plant proteins from a crude sample of N-sugar chains of a plant to be tested by using a Buffer Y Buffer solution, and remove pigment impurities in the total plant proteins by using methanol and acetone to obtain the plant proteins.
5. The method according to claim 1, wherein the method of enzyme digestion is to use trypsin or chymotrypsin to enzyme-cleave plant proteins to obtain plant polypeptide samples.
6. The method according to claim 1, wherein the N-sugar chain releasing method is to cleave the obtained plant polypeptide sample with PNGase A reagent to release plant N-sugar chains.
7. The method according to claim 1, wherein in the step (2), the plant N-sugar chain sample and the solution of the plant ditag N-sugar chain internal standard are mixed in a volume ratio of 1:1.
8. The method of claim 1, wherein in step (2), the mass spectrum comprises any one or more of matrix-assisted laser desorption ionization mass spectrometry, electrospray mass spectrometry, tandem mass spectrometry, multi-stage mass spectrometry.
9. The method for accurately and relatively quantitatively analyzing the N-sugar chain of the separated and purified plant and the application of the method in N-sugar group of the plant are as follows:
(1) Respectively obtaining relative quantitative data of N-sugar chains from different samples by the accurate relative quantitative analysis method of the plant N-sugar chains according to any one of claims 1 to 8;
(2) Comparing and analyzing relative quantitative data of the same N-sugar chains in all samples, wherein the same N-sugar chains refer to N-sugar chains with the same mass displacement and error within +/-0.4 in a detection map of all samples so as to meet the screening difference N-sugar chains with a significance difference p <0.05, and analyzing the effect of the difference N-sugar chains on the regulation and control of plant physiological processes.
10. Use of the method for the accurate relative quantitative analysis of plant N-sugar chains according to any one of claims 1-8 in fruit ripening studies.
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