CN109212227B - Product for screening and evaluating liver disease/cirrhosis related to saliva specific glycoprotein sugar chain structure and application - Google Patents
Product for screening and evaluating liver disease/cirrhosis related to saliva specific glycoprotein sugar chain structure and application Download PDFInfo
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Abstract
The invention discloses a product for screening and evaluating related liver diseases/cirrhosis based on saliva specific glycoprotein sugar chain structures and application thereof, and provides two products for screening and early diagnosis of liver diseases/cirrhosis, risk evaluation, drug screening and/or curative effect evaluation aiming at saliva samples, wherein one product detects the expression levels (whether high expression is available) of three specific glycoprotein sugar chain structures beta-D-GlcNAc, Gal beta 1-4GlcNAc and (GlcNAc beta 1-4) N in the saliva samples, and the other product detects whether any one or any combination of specific 11N-sugar chains is contained in the saliva samples. Correspondingly, the lectin with the specific binding recognition function is DSA, and can be independently used as a reagent for recognizing the sugar chain structure of the saliva specific glycoprotein to prepare related medical products.
Description
Technical Field
The invention relates to a method for determining a liver disease (especially liver cirrhosis) marker based on saliva-specific glycoprotein, and a related product and application thereof.
Background
Cirrhosis (HC) is a common chronic liver disease in our country, and is often seen as the prognosis of viral hepatitis and fatty liver disease. At present, various detection methods for cirrhosis are respectively limited by technical characteristics, and have the defects of low specificity, inconvenient sampling, incapability of early diagnosis and the like. Recent studies have shown that: with the development of liver diseases, abnormal changes in glycosylation of glycoproteins occur in liver tissues of patients and body fluids such as serum and saliva.
Patent document CN105675893A discloses a scheme for identifying hepatitis patients by detecting sugar chain markers based on serum/co-based on proteins in serum and saliva. Wherein:
based on serum detection, PTL-I, PNA, AAL, LTL, STL, BS-I, PTL-II, SBA, UEA-I, MAL-I, PHA-E + L are all up-regulated, while ACA is down-regulated, which can be distinguished from healthy people, i.e. suffering from liver disease; on the basis, when WFA, GSL-II, MAL-II, SJA, LEL, PWM and BPL are all up-regulated, and EEL is down-regulated, the population corresponding to the sample can be determined to have severe hepatitis B.
Based on the combined detection of serum and saliva, STL, SBA, WFA and GSL-II can be distinguished from healthy people when STL, SBA, WFA and GSL-II are all up-regulated and ACA is down-regulated, i.e. liver disease is caused.
The scheme needs more agglutinin when detecting a sample and needs combined statistics, so the method is not simple and effective for screening, diagnosing and evaluating the liver cirrhosis (HC).
It is important to note that some factors in blood can reach salivary glands with the circulation of body fluids, or enter the oral cavity with saliva secretion or cause changes in the transcriptional profile of genes in saliva, causing changes in the abundance or species of certain proteins, reflecting changes in the levels of some proteins in the blood. However, it has been found that 1939 proteins are present in human saliva and 3020 proteins are present in human plasma, and that only 27% of the saliva proteome coincides with the plasma proteome. Among the 177 potential biomarkers of proteins found to be associated with cardiovascular disease by plasma proteomics, only 40% of the proteins were also present in the salivary proteome; of the 1058 proteins listed as potential cancer-associated biomarkers, only 34% of the proteins were present in the salivary proteome. This indicates that many biomarkers in blood circulation are not identifiable in saliva. In addition, even the same protein is usually present in significantly lower amounts in saliva than in serum. Therefore, in practice, there is no necessary connection between the two, and researchers are often required to go through a lot of experiments and analyses to judge whether there is a possibility of proteome coincidence.
Thus, the above protocols have to give different protocols based on serum tests, respectively based on serum and saliva co-tests.
Disclosure of Invention
With regard to screening, diagnosis, evaluation, etc. of cirrhosis (HC), the inventors have determined the following conclusions through a number of experiments and analyses:
firstly, beta-D-GlcNAc, Gal beta 1-4GlcNAc and (GlcNAc beta 1-4) n sugar chain structures recognized by the lectin DSA are highly expressed (P is less than or equal to 0.0343) in saliva of patients with liver diseases (hepatitis B (HB), cirrhosis (HC) and liver cancer (HCC)) compared with sugar chain structures of other glycoproteins in saliva; and β -D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structures were expressed at the highest level in saliva of HC patients (p.ltoreq.0.0034) compared to Healthy Volunteers (HV), hepatitis B and liver cancer.
Two, the following 11N-sugar chains were present only in saliva of HC patients, but not in saliva of HV, HB, and HCC.
m/z.1996.7238(Fuc)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2006.7316(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2037.7503(Fuc)1(GalNAc)2(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2281.8321(Sia)1(Fuc)1(GalNAc)1(Gal)2(GlcNAc)2+(Man)3(GlcNAc)2；
m/z.2573.9480(Sia)1(Fuc)2(Gal)2(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2772.9572(Sia)2(Gal)4(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2794.0427(Fuc)3(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2(Fuc)1；
m/z.2806.9879(Fuc)1(Gal)6(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2924.0805(Gal)4(GlcNAc)6+(Man)3(GlcNAc)2(Fuc)1；
m/z.3232.1912(Fuc)2(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2；
m/z.3813.3617(Sia)3(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2)。
From the first conclusion above, the following application scenarios have been derived:
a product for screening, early diagnosis, risk assessment, drug screening and/or efficacy assessment of liver disease, the product comprising, for a saliva sample:
A. means for obtaining expression levels of three specific glycoprotein sugar chain structures, which are β -D-GlcNAc, Gal β 1-4GlcNAc, and (GlcNAc β 1-4) n, respectively;
B. a marker, a module or a processor for discriminating whether or not the sugar chain structure of the above three specific glycoproteins is highly expressed. The mark is used for displaying qualitative conclusion (existence, comparison and the like) in a form similar to a test strip, the module and the processor are used for displaying numerical quantification results, and the qualitative conclusion can be displayed and/or output; the specific implementation method belongs to the conventional technical means.
Taking drug screening and efficacy evaluation using the product as an example, the expression levels of the above two specific glycoprotein sugar chain structures at different time points before and after administration (course of treatment) to a subject can be determined, and if the expression levels are not reduced, the therapeutic effect of the drug is not good.
The above-mentioned apparatus comprises a lectin chip on which at least lectin DSA (lectins capable of specifically recognizing β -D-GlcNAc, Gal β 1-4GlcNAc, and (GlcNAc β 1-4) n sugar chain structures on glycoproteins) is disposed, an incubation cassette, and a biochip scanning system.
The above-mentioned identifier, module or processor is pre-recorded with a reference value corresponding to a Healthy Volunteer (HV) for determining whether high expression is present in comparison with the sample results. For example, a ratio of fluorescence signal values greater than 1.35 may be used to determine high expression.
Use of a lectin alone as a reagent for saliva-specific glycoprotein carbohydrate chain structure recognition, the lectin being DSA, for the preparation of a related product, being a kit, a device, an operable system and/or a combination thereof, for screening, early diagnosis, risk assessment, drug screening and/or efficacy assessment of liver diseases.
From the second conclusion above, the following application scenarios can be derived:
use of a recognition unit for a specific sugar chain for constructing a product for liver cirrhosis (HC) screening, early diagnosis, risk assessment, drug screening and/or efficacy assessment based on a saliva sample, said recognition unit recognizing any one or any combination of the following 11N-sugar chains (which can be concluded by judging "presence", "absence"):
m/z.1996.7238(Fuc)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2006.7316(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2037.7503(Fuc)1(GalNAc)2(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2281.8321(Sia)1(Fuc)1(GalNAc)1(Gal)2(GlcNAc)2+(Man)3(GlcNAc)2；
m/z.2573.9480(Sia)1(Fuc)2(Gal)2(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2772.9572(Sia)2(Gal)4(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2794.0427(Fuc)3(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2(Fuc)1；
m/z.2806.9879(Fuc)1(Gal)6(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2924.0805(Gal)4(GlcNAc)6+(Man)3(GlcNAc)2(Fuc)1；
m/z.3232.1912(Fuc)2(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2；
m/z.3813.3617(Sia)3(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2。
a product for cirrhosis (HC) screening, early diagnosis, risk assessment, drug screening and/or efficacy assessment, the product comprising, for a saliva sample:
A. lectin DSA;
B. an affinity chromatography solid phase support for coupling lectin DSA;
C. a reagent and a device for separating N-sugar chains from glycoproteins having a β -D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) N sugar chain structure;
D. a mass spectrometer for displaying m/z values corresponding to the respective N-sugar chains;
E. a label, a module or a processor for identifying the 11 rock N-sugar chains according to a mass spectrogram so as to obtain a judgment result of whether any one or any combination of the 11 rock N-sugar chains is contained.
The affinity chromatography solid phase carrier for coupling lectin DSA can be selected from Fe3O4Magnetic microparticles, agarose gels, sephadex, glass microspheres, and the like.
Drawings
Fig. 1 shows a boxplot analysis of lectin DSA results, in which 215 saliva samples (HV, n-51; HB, n-51; HC, n-68; HCC, n-45) were tested in one case. In the figure, the ordinate represents the normalized fluorescence intensity NFI corresponding to the lectin on the lectin chip, and each horizontal line from top to bottom in each box represents the maximum value, the last quartile, the median, the first quartile, and the minimum value of the group of data. The horizontal line in the graph represents the comparison between the two groups shown at both ends, and P is the P-Value obtained from the one-way analysis of variance, where P <0.05 indicates significant difference, P <0.01 indicates very significant difference, and P <0.001 indicates very significant difference. HV: saliva of healthy volunteers, HB: saliva of hepatitis b patient, HC: cirrhosis patient saliva, HCC: saliva of liver cancer patients.
FIG. 2 is a scattergram of DSA intensity of binding of each probe of the saliva chip. The horizontal line in the graph represents the comparison between the two groups shown at the two ends, and P is P-Value obtained from one-way variance analysis, where P <0.05 indicates significant difference, P <0.01 indicates very significant difference, and P <0.001 indicates very significant difference. HV: saliva of healthy volunteers, HB: saliva of hepatitis b patient, HC: cirrhosis patient saliva, HCC: saliva for liver cancer patients
FIG. 3 shows the results of SDS-PAGE silver staining and DSA blotting experiments on four groups of mixed saliva samples, in which: a is a result graph of silver staining color development; b is a graph of the results obtained by scanning at 635nm after DSA lectin blotting. The sample information of each band is: 1: protein Ladder, 2: saliva of healthy volunteers, 3: saliva of hepatitis b patient, 4: cirrhosis patient saliva, 5: saliva of liver cancer patients.
Detailed Description
The following detailed description is provided for the verification experiments and analysis of the present application, and the specific development process of the inventors is not limited thereto.
Screening of saliva protein differential sugar chain structure of patients with chronic hepatitis B, liver cirrhosis and liver cancer
The research method comprises the following steps:
1.1 saliva sample Collection and pretreatment
Saliva samples of healthy volunteers and patients with chronic hepatitis B, liver cirrhosis and liver cancer adopted in the experiment are strictly subject to ethical examination and approval (Human Research Ethics Committees (HRECs)) of northwest university, fourth department of medical sciences, Tang-city hospital, Shanxi province people hospital and second subsidiary hospital of the Western-Ann transportation university. All volunteers donated saliva samples, along with clinicians assisting in sampling guidance, were informed, consented and highly coordinated to the study work, completing collection of saliva samples under uniform sampling requirements. The concrete requirements are as follows: the sample donor needs to be free from diabetes, other organs except the liver should be free from chronic diseases such as inflammation and tumor, the donor needs to be determined not to eat within 3 hours before saliva is collected and not to take medicines within 24 hours when sampling, then the donor needs to rinse the mouth three times by using clean sterile physiological saline (0.9% NaCl) to ensure the oral hygiene of the donor and no food residues, the tongue tip of the donor is propped against the palate and the saliva sample naturally secreted under the tongue is collected into a 2mL centrifuge tube, and 10 mu L Protease Inhibitor (Protease Inhibitor Cocktail, Sigma-Aldrich, U.S. A) is immediately added and temporarily stored in ice bath. A total of 215 saliva samples were collected under clinician guidance: among them, 51 healthy volunteers (HV, n ═ 51), 51 patients with chronic hepatitis caused by hepatitis b (HB, n ═ 51), 68 patients with chronic cirrhosis (HC, n ═ 68), and 45 patients with liver cancer (HCC, n ═ 45). Specific sample information is shown in table 1.
Collecting saliva within 12 hours, subpackaging saliva into centrifuge tubes according to 1mL, adding 1 XPBS to complement to 1mL if the quantity is less than 1mL, centrifuging for 10,000g × 15min, carefully sucking supernatant, measuring the concentration by a micro nucleic acid protein analyzer (Nano-drop), adding protease inhibitor according to the quantity of 1mg saliva protein to 10 μ L protease inhibitor, mixing uniformly, and subpackaging at-80 ℃ for storage.
TABLE 1 saliva sample information for small-Scale lectin chip case-by-case detection for liver disease diagnosis
1.2 fluorescent labeling and quantitation of Individual salivary proteins
Taking 100 μ g of saliva sample quantified by Nano-drop, adding 0.1mol/L Na in equal volume2CO3/NaHCO3pH9.3 buffer, 1mg: 120. mu.L of Cy3 fluorescent dry powder was dissolved in DMSO and 5. mu.L of fluorescent solution was added to the sample and incubated at room temperature for 3 hours, during which the sample was kept strictly protected from light and kept shaking. After the reaction is finished, adding 10 mu L of 4M hydroxylamine solution into the sample, reacting for 5 minutes on ice, and after excessive free fluorescence is fully blocked, carrying out gel column chromatography on the protein sample by utilizing a Sephadex G-25 molecular sieveSeparation is carried out. And (3) collecting the fluorescent sample by using a new 1.5mL centrifuge tube, quantifying, protecting the fluorescence-labeled sample from light to prevent the quenching of the fluorescent group, and storing at-20 ℃.
1.3 lectin chip example by example detection of differences in salivary protein sugar chain expression
The experiment utilizes a 14-probe small-scale lectin chip self-made by a laboratory, and specifically comprises lectins MAL-II, UEA-I, PTL-I, LTL, RCA120, DSA, GNA, PHA-E + L, EEL, AAL, STL, ACA, WGA and MAL-I. After a 75X 25mm glass slide with high specification, super clean and microscopically smooth surface is cleaned by a decontamination lotion and strong acid, the surface of the glass slide is modified by epoxy silane by using a (3-Glycidyloxypropyl) trithioxysilane reagent, so that the surface of the glass slide is provided with a couplable lectin-NH2Epoxy groups of (a). A Smart Arrayer 48 sample applicator is used for quantitatively sampling negative quality control (BSA), an internal reference probe PTL-II, 14 detection probes and positive quality control (Cy3 fluorescent labeled BSA), each quality control or probe is sampled in parallel for 3 points to prepare a 9 x 7 (point spacing 850 mu m) lectin microarray chip, and the lectin microarray chip is stored at 4 ℃ after sample application. 4 arrays are prepared on each piece of epoxy slide, the array covering surface ensures that the array can be matched with the adhesive tape sealing area of the chip reaction box, and accordingly, each chip can simultaneously detect 4 different protein samples.
Before the lectin chip assay samples were performed, the chip was removed from 4 ℃, vacuum-warmed at 37 ℃ for 30min, then washed 5min × 2 times with 1 × PBST on a horizontal shaking shaker at 70rpm, and then washed 5min × 2 times with 1 × PBS to sufficiently wash free lectin not coupled to the slide. After washing, the residual PBS was spun off using a small glass slide centrifuge. Before loading, 120 μ L of lectin chip blocking solution was added to each array region of the incubation box to block the epoxy groups on the blank surface of the slide to reduce the signal value on the back of the slide during fluorescence scanning. After the incubation box is sealed, the reaction is carried out for 1h at 37 ℃ in a constant-temperature incubation box, after the sealing is finished, 1 XPBST is cleaned for 5min multiplied by 2 times, 1 XPBS is cleaned for 5min multiplied by 2 times, after the drying is carried out, protein sample incubation systems (80 mu L agglutinin chip incubation liquid, 8 mu L4M hydroxylamine, 2 mu L0.1% Tween-20, 4 mu g Cy3 labeled saliva protein samples and ultrapure water are added into each array area of the incubation box to complement the final volume to 120 mu L) are added into the array areas of the incubation box, the incubation is carried out for 3h at 37 ℃, after the reaction is finished, 1 XPBST is cleaned for 5min multiplied by 2 times, 1 XPBS is cleaned for 5min multiplied by 2 times, the drying is carried out. The final data reading of the lectin chip is realized by using a Genepix 4000B chip scanner manufactured by Axon, and the scanning parameters are set as follows: excitation wavelength 532nm, PMT power 70% and excitation intensity 100%.
1.4 lectin chip data analysis
The quantification process of the lectin chip fluorescence signal was performed by GenePix Pro (4000B) software, and data obtained by data extraction for each array included: the net difference FI (fluorescence intensity) obtained by subtracting the background signal from the probe signal, the standard deviation SD (Standard development) of the background, and the like. In the analysis process, firstly, judging the validity of the point data according to the FI/SD of each point, taking the point with the FI/SD being more than or equal to 1.5 as valid data, calculating the standard normalized fluorescence signal value NFI (normalized fluorescence intensity) of each probe, namely dividing the mean FI value of each probe by the sum of the FI values of 14 detection probes, and expressing the sum as follows by using a formula: NFIx＝Median FIx/(Median FI1+Median FI2+Median FI3+…+Median FI14) From this, NFI corresponding to 14 lectin probes on each array were obtained and used for statistical analysis.
The statistical Analysis is mainly to perform One-factor Analysis of variance (One-Way ANOVA Analysis) among groups on four groups of data of HV, HB, HC and HCC by using GraphPad Prism 6.0, and the specific method is to record the data into a "Column" Analysis program according to groups, and select a Box diagram "Box & Shiskers" or a Scatter diagram "Scatter & SD" and the like according to the drawing requirements. The "Analyze" analysis program was then entered to compare pairwise across groups and report the significance of the difference, P-Value.
The research results are as follows:
2.1 lectin chip analysis of differences in salivary glycoprotein sugar chain expression in liver disease patients
215 saliva samples were tested by example using the lectin chip array shown in fig. 1, and each NFI value was obtained by chip data processing, and the average NFI result calculated from each set of data is shown in table 2. The mean NFI results visually indicate the binding of each group of saliva samples to each probe, and the dispersion of each lectin from each sample can be assessed according to its standard deviation SD. In addition to the experimental data, the table also enumerates the sugar chain types specifically recognized by each lectin probe, such as Sia α 2-3Gal, Sia α 2-3GalNAc sialylated sugar chain structures recognized by lectin MAL-II, Man α 1-3Man polymannan sugar chain structures recognized by GNA, and the like.
TABLE 2 test results of the lectin chip
aLectin Probe, 14 Lectin probes contained in the chip array;bspecific to Glycopeterns, glycoforms specifically recognized by each lectin (Glc, glucose; GlcNAc, acetylglucosamine; Gal, galactose; GalNAc, acetylgalactosamine; Man, mannose; Sia, sialic acid; Fuc, fucose);cNFI, normalized fluorescence intensity for each probe after chip data normalization, and data in the table show the mean NFI of the sample and its standard deviation SD.
2.2 screening of sugar chains of salivary differential glycoproteins from liver cancer patients
The Ratio values between the respective phase groups were obtained by pairwise comparisons between the groups using the respective average NFI values listed in table 2, and it was defined that Ratio >1.500 represents that sugar chains recognized by the lectin exhibit high expression in the comparison between the groups, and Ratio <0.667 represents that sugar chains recognized by the lectin exhibit low expression in the comparison between the groups. After comparison among groups, the sugar chains recognized by the lectins MAL-II, AAL, STL, ACA, WGA, MAL-I were found to have no expression difference among the comparisons among groups. Additional 8 different lectins (UEA-I, PTL-I, LTL, RCA120, DSA, GNA, PHA-E + L, EEL) from group to group and detailed data are shown in Table 3. The analysis found that the lectins PTL-I recognizing GalNAc α -1,3Gal and GalNAc α -1,3Gal β -1,3/4Glc structure had high expression of NFI on average in HCC group compared to HV and HB, while there was no difference in the comparison between HCC and HC; compared with HB, the lectin EEL recognizing the Gal alpha 1-3(Fuc alpha 1-2) Gal structure has high expression of NFI on average in HCC group, and HCC has no difference compared with HV and HC; lectin LTL recognizing Fuc α 1-2Gal β 1-4GlcNAc and Fuc α 1-3(Gal β 1-4) GlcNAc structures exhibited high expression of NFI on average in the HCC group, compared to HV, HB, and HC. The differential analysis of the mean NFI showed that these 3 lectin-recognized sugar chains had a tendency to be relatively highly expressed in the saliva of liver cancer patients.
TABLE 3 Difference analysis of mean NFI of four groups of saliva assays
The table lists the Ratio values calculated by comparing four groups of average NFI of HV, HB, HC and HCC two by two, and defines Ratio A/B >1.500 to indicate that when the expression level of a specific sugar chain structure recognized by a certain lectin is compared between A, B groups, group A is highly expressed than group B, and an upward arrow is marked in the table; ratio a/B <0.667 indicates that when the expression level of a specific sugar chain structure recognized by a lectin was compared between A, B groups, group a was expressed lower than group B, and the downward arrow is marked in the table.
The statistical analysis of individual data corresponding to the lectin DSA probe was used to determine the significance of differences in the sugar chain structures identified by lectin DSA in the saliva of cirrhosis patients by one-way anova, and as a result, a series of statistical data was shown in fig. 1, in which the significance of differences and the degree of dispersion compared between groups was visually reflected. According to the result of the one-way anova between groups: the beta-D-GlcNAc, Gal beta 1-4GlcNAc and (GlcNAc beta 1-4) n sugar chain structures recognized by the lectin DSA are highly expressed (P. times. ltoreq. 0.0343) in saliva of patients with liver disease (hepatitis B, cirrhosis, liver cancer) as compared with other glycoprotein sugar chain structures in saliva; and β -D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structures were most highly expressed in saliva of HC patients (p ^ 0.0034) compared to Healthy Volunteers (HV), Hepatitis B (HB) and liver cancer (HCC). And this result is substantially consistent with the result of the average NFI comparison.
Therefore, it can be finally found that: compared with other glycoproteins, the beta-D-GlcNAc, Gal beta 1-4GlcNAc and (GlcNAc beta 1-4) n sugar chain structures identified by the lectin DSA are highly expressed in saliva of patients with HB, HC and HCC, and the beta-D-GlcNAc, Gal beta 1-4GlcNAc and (GlcNAc beta 1-4) n sugar chain structures identified by the lectin DSA are highly expressed in the saliva of patients with HC (p is less than or equal to 0.0034) compared with HV, HB and HCC. This can therefore be considered as a basis for cirrhosis (HC) screening, early diagnosis, risk assessment, drug screening and/or efficacy assessment.
Second, the isolation and sugar chain identification of saliva specific glycoprotein by using lectin DSA for healthy volunteers and patients with hepatitis B, liver cirrhosis and liver cancer
The research method comprises the following steps:
up-regulated expression of the sugar chain structures of beta-D-GlcNAc, Gal beta 1-4GlcNAc and (GlcNAc beta 1-4) n recognized by DSA in the HC group was verified using 90 saliva samples of patients with chronic liver disease (HB, n-30; HC, n-30; HCC, n-30) and 30 saliva samples of healthy volunteers strictly conforming to the typical characteristics of each stage. By preparing saliva chips from 120 samples of saliva, the abundance of the sugar chain structures of β -D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) n in each patient's saliva on the chips was examined by lectin DSA and the differences were analyzed by comparison. In addition, mixed saliva samples of each group were obtained by equal mass mixing, and differences in the abundance of sugar chain structures of β -D-GlcNAc, Gal β 1-4GlcNAc, and (GlcNAc β 1-4) n in the mixed saliva samples of each group were verified by SDS-PAGE protein denaturation electrophoresis and Lectin Blotting (Lectin Blotting). After the series of verification, a specific glycoprotein in each group of saliva mixed samples is enriched by using a Magnetic Particle composite (DSA-Magnetic Particle composites, LMPCs) prepared by coupling agglutinin DSA on the surface of a ferroferric oxide nano Magnetic Particle, N-sugar chains and O-sugar chains on the specific glycoprotein are separated by using a PNGase F enzyme cutting method and a NaClO oxidation method, Mass Spectrum identification is carried out on the sugar chains by using Matrix-Assisted Laser Ionization Flight Time Mass Spectrum (Matrix-Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrum, MALDI-TOF-MS), and sugar chain structures are conjectured, so that a healthy volunteer identified by the agglutinin DSA and a saliva specific glycoprotein sugar chain Spectrum of a hepatitis B patient, a liver cirrhosis patient and a liver cancer patient are obtained and contrastively analyzed for differences, and the differences are specifically as follows.
3.1 selection, mixing and quantification of saliva samples
Samples collected in the experiment all come from the fourth university of military medical science, Tang university Hospital, and volunteers who provide the samples all have the five-index test of hepatitis B and the liver function test data, and the samples are grouped according to the guide of the sampling clinicians. Statistically, statistical and clinical threshold references were taken for the following assay data: hepatitis b surface antigen HBsAg; hepatitis b e antigen HBeAg; hepatitis b core antibody hbcabs; alanine aminotransferase ALT (normal reference value: 0-40U/L); aspartate aminotransferase AST (normal reference value: 0-40U/L); glutamyl transpeptidase GGT (normal reference value: 0-50U/L); albumin (normal reference value: 35-55 g/L); alpha-fetoprotein AFP (normal reference value: 0-200 ng/mL). In addition, statistics were made for age and gender, and the statistical information is shown in table 4.
Table 4 saliva sample information statistics for validation and mass spectrometric identification
Saliva sample collection and processing methods 120 saliva samples listed in the table were quantified using a micro nucleic acid protein analyzer, as described above, and then mixed by weighing 500. mu.g of each saliva sample by mass. Four saliva mixed samples of Healthy Volunteers (HV), chronic Hepatitis B (HB), chronic cirrhosis (HC) and liver cancer (HCC) are obtained. The concentration of the mixed sample was determined using a BCA protein quantification kit (bio-cloud biotechnology, shanghai, china).
3.2 saliva chip design and sugar chain glycoform differential verification
The experiment designs a saliva chip which takes 30 saliva samples of healthy volunteers and patients with chronic hepatitis, liver cirrhosis and liver cancer as reaction probes, the total number of the saliva probes is 120, 4 negative quality controls and 4 positive quality controls are added, and each sample is spotted on the chip for 3 continuous repetitions to prepare a saliva microarray chip with the size of 12 multiplied by 32 (the dot spacing is 1 mm). When the expression abundance of the sugar chain structures of beta-D-GlcNAc, Gal beta 1-4GlcNAc and (GlcNAc beta 1-4) n in each saliva sample on the saliva chip was relatively quantitatively determined by using lectin DSA, a Cy5 fluorescent dye was used to label lectin DSA and a reaction system (400. mu.L of lectin chip incubation liquid, 40. mu.L of 4M hydroxylamine, 10. mu.L of 0.1% Tween-20, 20. mu.g of Cy 5-labeled lectin DSA, and the final volume of ultrapure water was made up to 600. mu.L) was prepared and applied to the saliva chip. After the combination reaction of the saliva chip and Cy5-DSA is completed and cleaned, data acquisition is finally completed on a Genepix 4000B chip scanner (parameters are set to be 635nm for excitation wavelength, 70% for PMT power and 100% for excitation intensity).
After obtaining the NFI value for each probe, the data with SD <1.5 was rejected by examining the Standard Deviation (SD) of each data provided by the scanning system, then taking the Median values of 3 replicate spots per probe, making scatter plots of the Median value data of each saliva sample in GraphPad Prism 6.0 software as four sets of HV, HB, HC and HCC and statistically analyzing by the origin One-Way ANOVA Analysis.
3.3 Polyacrylamide gel electrophoresis (SDS-PAGE) and band visualization
The SDA-PAGE gels were transferred to an electrophoresis chamber and the direction of the gel slide and the direction of the electrodes were checked to ensure that the current was in the on-state. In this study, the protein samples separated by SDS-PAGE need to identify the target bands on the gel by two chromogenic methods, i.e., Silver oxide Staining (Silver Staining) is used to compare the differences of total proteins and lectin blotting is used to verify the differences of sugar chain expression of specific glycoproteins identified by DSA. The Loading amount of the saliva protein sample for silver staining is 8 mug, the Loading amount of the saliva protein sample for Lectin Blotting is 30 mug, each group of samples with equal mass are taken according to the experiment requirement, 4 mug of 5 Xloading Buffer is added, 20 mug of ultrapure water is used for supplementing, the samples are placed in boiling water for 5min after shaking and uniform mixing, and the samples are quickly centrifuged after water bath and cooled on ice for Loading. The samples were sequentially applied to each gel well in the order of Protein Ladder, HV, HB, HC, and HCC. The electrophoresis parameters were set as: under the 80V constant voltage mode, when the Loading Buffer forms a slender and horizontal straight line in the concentrated gel layer, the power supply is adjusted to the 110V constant voltage mode until the electrophoresis is finished.
Silver staining and color development: SDS-PAGE gel for silver staining should be taken out from the gel plate in time after the electrophoresis reaction is finished, and soaked in stationary liquid (50% ethanol, 10% glacial acetic acid, water) for more than 2 h. After fixing until the gel volume is reduced to half, the gel is reacted in a soak solution (30% ethanol, 6.8% sodium acetate pentahydrate, 0.2% sodium thiosulfate, 0.15% glutaraldehyde, water) for 30 min. Soaking in ultrapure water for swelling again after the reaction of the soaking solution, and soaking for 20min × 3 times after water change. The gel was soaked with staining solution (0.5% silver nitrate, 0.1% formaldehyde, water) in order to bind silver ions to the proteins in the gel. All the reaction processes are vibrated at low speed on a shaking table, and the reaction liquid and the gel in each step are kept to be uniformly contacted. After dyeing reaction for 20min, the gel and the reaction vessel were rinsed with ultrapure water for 1min, and then color developing solution (2.5% sodium carbonate, 0.1% formaldehyde, water) was poured in. Stopping the color development of the strips in the gel to a proper degree by using a stop solution (1 percent glacial acetic acid and water) in time and collecting images, and strictly using ultrapure water in the whole process to avoid introducing Cl into the solution in each step-、CO3 2-、SO4 2-The plasma affects the color reaction.
Lectin blotting experiments: the SDS-PAGE gel for the blotting experiment is taken out of the gel plate after the electrophoresis reaction is finished, the redundant part is cut off and soaked in the 1 Xrotary membrane buffer solution for 10min, 4 pieces of filter paper with the same area as the gel and 1 piece of PVDF blotting membrane which is activated for 30s by soaking in methanol are soaked together with the SDS-PAGE gel. From the positive to the negative of the electrophoresis tank channel, the blotting system should be: and (3) sequentially discharging 2 pieces of filter paper, a PVDF blotting membrane, gel and 2 pieces of filter paper, placing the materials in an electrophoresis tank after ensuring that the materials are tightly attached without bubbles, and setting parameters to perform constant current electrophoresis for 1h 45min under 300mA to complete the blotting reaction. After the reaction is finished, taking out the imprinted PVDF membrane, and putting the membrane in Ca diluted to working concentration to avoid pollution and physical damage in the processThe adsorption of nonspecific Free polysaccharide on the membrane was removed in a rbo-Free Blocking solution for 1 h. After blocking, the PVDF membrane was transferred to 1 XTSS, Cy5-DSA was added to a final concentration of 5. mu.g/mL and Ca was added to a final concentration of 0.1mM2+Shaking at 4 deg.C in dark for 12 h. After the reaction is finished, washing with 1 × TBST for 15min × 3 times, and the whole washing process should be strictly protected from light to prevent fluorescence quenching. Scanning fluorescence signal images by using a Storm 840 multifunctional image analyzer, and setting PMT to be 800, resolution 100DPI and excitation light wavelength to be 635nm according to parameters.
3.4 preparation of lectin-magnetic microparticle complexes and magnetic separation of the corresponding glycoproteins
This experiment utilized a laboratory prepared epoxidized Fe3O4Magnetic particles are coupled with lectin DSA to prepare a lectin Magnetic Particle complex (DSA-Magnetic Particle complexes, LMPCs), and then the specific glycoprotein which is affinity-bound by the lectin DSA is obtained by mixing, magnetically separating, washing and eluting the LMPCs and a sample. Firstly, taking epoxidized Fe3O43mg of magnetic particles (the material is obtained by a co-immunoprecipitation method and modified by epoxidation, see the literature for a specific method[46]) Adding 1mL of coupling buffer solution, repeatedly reversing until the coupling buffer solution is fully mixed, placing the mixture on a magnetic separator, and discarding supernatant liquid to realize cleaning when the magnetic particles are completely adsorbed to a magnet, wherein the cleaning step is multiplied by 3 times. Then, 300 μ g of DSA agglutinin is dissolved by 600 μ L of coupling buffer solution and added into the magnetic particle system for re-suspension and uniform mixing, and the mixture is placed on a horizontal shaking table for reaction at 180rpm and 25 ℃ for 6 hours to complete the coupling of the agglutinin DSA and the magnetic particles. After the coupling is finished, carrying out magnetic separation on a magnetic separator while discarding supernatant, adding 1mL of magnetic particle confining liquid, washing the LMPCs through the steps of resuspending, oscillating, magnetically separating and discarding supernatant, repeating the washing for 3 times, adding 600 mu L of magnetic particle confining liquid, and reacting on a shaking table for 1h to confine redundant epoxy groups. After the blocking was completed, the LMPCs were washed 3 times with the binding buffer solution, while the saliva sample 2mg was dissolved with a volume of 600. mu.L of the binding buffer solution (if the original concentration of saliva is too small, after concentration by centrifugal ultrafiltration at 14,000 Xg using a 10kDa ultrafiltration centrifuge tube (Millipore Corp, U.S.A.) after filling the corresponding mass sampleThe sample is then added into an LMPCs system for resuspension after being subjected to volume fixing to 600 mu L by using a combined buffer solution, and the sample is shaken at 180rpm and reacted at 25 ℃ for 3 hours. The washing step as described above was repeated at least 5 times using a washing buffer solution before elution. And (3) determining the protein concentration in the cleaning solution in real time by using the Nano-Drop, and when the protein concentration in the cleaning solution is detected to be 0, indicating that the non-specifically bound protein is completely separated. Adding 400 μ L elution buffer, shaking on shaking table at 180rpm for 20min, collecting supernatant after magnetic separation, dissolving specific glycoprotein in the supernatant due to lectin denaturation, adding 200 μ L elution buffer, repeating the elution step once, and collecting supernatant.
3.5 enzymatic isolation of glycoprotein N-sugar chains (N-Glycans)
Adding 200 μ g of specific glycoprotein separated from LMPCs into a 10KDa ultrafiltration tube (Millipore Corp, U.S. A), adding 8M urea to the maximum liquid carrying capacity scale in the ultrafiltration tube, centrifuging for 10min at 14,000 Xg, and filtering inorganic salt ions and non-protein micromolecules along with solvent through the ultrafiltration membrane into a collection sleeve; adding 400 mu L of 8M urea, uniformly mixing, centrifuging, and repeating the step for 3 times to fully denature the protein sample; adding diluted 1 XDTT working solution 400 μ L, fully blowing, sucking, mixing, reacting at 56 deg.C for 45min, and centrifuging at 14,000 Xg for 10 min; adding 400 μ L of 1 × IAM working solution into the tube, and reacting at room temperature in dark for 30min to seal the disulfide bond; 400 μ L of 40mmol/L NH was added4HCO3Centrifuging to remove residual reaction solution of each step and repeating the step for 3 times; adding 20 mu L of Trypsin protease preactivated by hydrochloric acid, and carrying out enzyme digestion at 37 ℃ overnight; after the reaction is finished, boiling water bath is carried out for 1min to inactivate Trypsin protease, then a new collecting sleeve is replaced for the ultrafiltration membrane, 2 mu L PNGase-F glycosidase is added into the ultrafiltration membrane, 14,000 Xg centrifugation is carried out for 10min after 10h reaction, 400 mu L40 mmol/L NH is added4HCO3Repeating the centrifugation for one time, filtering the N-sugar chain into a new collecting sleeve after the centrifugation for two times, and carrying out vacuum centrifugation and freeze-drying on the N-sugar chain solution on a centrifugal concentrator at the temperature of-20 ℃.
3.6NaClO Oxidation for O-sugar chain (O-Glycans)
Adding 200 mu g of glycoprotein obtained by LMPCs separation into a 10KDa ultrafiltration tube, centrifuging for 10min at 14,000 Xg to remove an original solvent phase, adding 400 mu L of ultrapure water, uniformly mixing, centrifuging, repeating the cleaning for at least 7 times, wherein the NaClO has strict requirements on pH in subsequent reactions, so that the quality of the ultrapure water and the thorough degree of the cleaning are particularly paid attention to in the experimental process; after the last cleaning, sequentially adding 200 mu L of ultrapure water and 100 mu L of 6% NaClO, and placing the mixture on a horizontal shaking table to react for 30min at room temperature; after the reaction is finished, ice bath precooling is carried out, 15 mu L of 10% formic acid (precooling preparation on ice in advance) is added to stop the oxidation reaction of the sodium hypochlorite, and in order to avoid over violent reaction, the reaction system is kept in the ice bath for 10 min; adding 400 mu L of ultrapure water for repeatedly cleaning for at least 7 times after centrifuging so as to fully remove the residual formic acid in the previous step; after the final washing, about 30. mu.L of solution should remain in the filter membrane at this time through centrifugation, 170. mu.L of ultrapure water is added into the filter membrane, the pH is adjusted to about 7.6 by formic acid, then 6.64. mu.L of 6% NaClO is added, a new collection sleeve is replaced, and the system is sealed by a sealing film and reacted for 24 hours at room temperature. After the reaction is finished, 8 mu L of precooled 10% formic acid is added to stop the reaction, 200 mu L of ultrapure water is added after centrifugation, the mixture is mixed and centrifuged, the O-sugar chain is fully filtered into a new collecting pipe after two times of centrifugation, and the O-sugar chain solution is subjected to vacuum centrifugation and freeze drying at the temperature of 20 ℃ below zero on a centrifugal concentrator.
3.7 gel desalting of sugar chains
Washing and nonpolar equilibration of Sepharose CL-4B: adding 100 mu L of Sepharose CL-4B gel into a 1.5mL enzyme-free centrifuge tube, adding 800 mu L of methanol water eluent, carrying out 9,000 Xg centrifugation for 5min after re-suspension and uniform mixing, taking out from the centrifuge, standing vertically in a centrifuge tube pore plate for 30s, carefully sucking and discarding supernatant liquid by a pipette after the gel plane is horizontal, and repeating the steps for 5 times by using methanol water eluent; adding n-butanol cleaning solution, repeating centrifugation, and discarding supernatant for 2 times to obtain gel to be loaded.
Sugar chain loading and desalting: taking 500 mu L of N-butanol cleaning solution to redissolve the N/O-glycosylated sugar chain sample after freeze-drying, then loading the sample into Sepharose CL-4B gel which is well balanced in advance, oscillating and reacting for 1h at 80rpm on a horizontal oscillation shaking table, and according to the polarity difference of solid phase and liquid phase, the polar sugar chain can be dredged in the liquid phase and combined into gel particles; after the sugar chain is combined with the gel, centrifuging for 5min at 9,000 Xg, removing the supernatant by a pipette, then adding 500 mu L of n-butanol cleaning solution, mixing uniformly, centrifuging, removing the supernatant, and repeating the cleaning step for 5 times to remove the nonpolar polypeptide and the salt ions in the sample; after the completion of the washing, 500. mu.L of methanol-water eluent was added thereto, and the mixture was reacted on a shaker at 180rpm for 20 minutes while the sugar chains were redissolved in the eluent due to the polarity of the methanol solution, centrifuged at 9,000 Xg after the completion of the reaction and the supernatant was collected by a new enzyme-free tube, and then eluted 1 time with 500. mu.L of methanol-water eluent. The purified N/O-sugar chain solution is freeze-dried by vacuum centrifugation at-20 ℃ on a centrifugal concentrator.
MALDI-TOF-MS Mass Spectrometry analysis of 3.8N-/O-sugar chain
Using 5 μ L of 1:1 methanol: and (v) re-dissolving the freeze-dried sugar chain crystals in the aqueous solution (v: v), uniformly blowing and sucking to ensure that a trace amount of sugar chain samples at the tube bottom are fully dissolved, then loading 2 mu L of sugar chain samples onto sample holes of a Bruker MTP Anchor chip 384 point target plate, observing the shape of the sample crystals on the stainless steel target plate after the samples are naturally air-dried and crystallized, wherein the crystal forms formed by the sugar chain samples under the proper concentration are supposed to be outwards radiated in a star shape in the range of circular liquid drops. Then 2. mu.L of DHB matrix is added in the range of the original sample crystallization, natural air drying is waited for recrystallization, the MTP Anchorchip 384-point target plate loaded with sugar chain crystallization is loaded on MALDI-TOF/TOF-MS (Bruker Daltonic, Germany), and the original sample is analyzed by primary mass spectrometry in TOF-MS mode, and the parameters are selected as follows: under the polysaccharide measurement spectrum of "RP-700 + 4000_ Da.Par", the resolution accuracy in the range is calibrated according to the standard, and the N-glycosylated sugar chain (1200 + 4000Da) and the O-glycosylated sugar chain (500 + 1800Da) are sequentially resolved according to the recorded target positions.
3.9 Mass spectrometric data analysis
And opening MASS Spectrum data with ' Ref ' as a suffix by utilizing analysis software flexAnalysis, setting a spectral peak signal-to-noise ratio (S/N) ≥ 3 as an automatic screening condition, reading data of each peak, and manually adding an individual higher peak into a data list through ' + (Find MASS Spectrum) in a software interface if the individual visible peak is not read due to noise influence. Through the above-mentioned series of operations, the "m/z.", "S/N", "Quality Fac", "Res" can be obtained6 items of digitized data were used, including "Intens." and "Area". Under the condition that the signal to noise ratio is effective (S/N is more than or equal to 3), selecting m/z. and Intens of each peak to enumerate and generate a text document, importing the text document into glycoform test software and automatically analyzing a sugar chain structure, wherein analysis parameters are set as: selecting a Glycome DB database and selecting an ion channel as "[ M + Na]+"and" [ M + H]+", charge is at most +1, precursor ion tolerance is + -1 Da, and fragment ion tolerance is + -0.5 Da.
The research results are as follows:
4.1 verification of DSA recognition of differences in sugar type of salivary sugar chains by saliva chips
120 of the saliva samples quantified competed for binding to lectin DSA in the same incubation environment, and the expression abundance of each of the β -D-GlcNAc, Gal β 1-4GlcNAc, and (GlcNAc β 1-4) n sugar chain structures was reflected by NFI at each saliva probe point. The Blood Group-H Antigen and T-Antigen structures recognized by lectin PTL-II were stably expressed in HV, HB, HC and HCC without difference. Using Cy5-PTL-II to react with saliva chip, the chip scanning results show: the sample application of each saliva probe on the chip is uniform, the negative quality control and the positive quality control have good indication effects, and the internal reference lectin PTL-II after sample loading shows that the sample probe has good quantitative uniformity and no experimental error caused by inaccurate quantification. Using Cy5-DSA to react with saliva chips, scattergrams were plotted from HV, HB, HC, and HCC groups based on chip scan results using Median values for each saliva probe FI, as shown in FIG. 2.
Statistical Analysis of the Analysis by orthodyne One-Way ANOVA Analysis among groups of HV, HB, HC and HCC (see table 5) revealed that β -D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structures recognized by lectin DSA were significantly increased in the groups of HB (p ═ 0.0021), HC (p ═ 0.0001) and HCC (p ═ 0.00320) as compared to the HV group. It is noted that the sugar chain structure specifically recognized by lectin DSA was significantly highly expressed in HC group, and significantly increased compared to HV group p ═ 0.0001), HB group (p ═ 0.0216) and HCC group (p ═ 0.0013)
TABLE 5 between groups Krustal-Wallis test and One-Way ANOVA analysis of four groups of samples
aMultiple comparison (1vs.2), grouping by pairwise comparison between four sets of samples;bmean rank difference, the inter-group Mean rating difference obtained by Krustal-Wallis test, (Mean rank 2-Mean rank 1);csignificant, significance of difference, Mean rank diff among groups>20 and p<Yes at 0.05, and No at the opposite.
4.2 lectin blot validation Mixed sample results
After the saliva samples of three groups of patients with chronic liver diseases and healthy volunteers were mixed to eliminate individual differences in the groups, the mixed samples were subjected to SDS-PAGE and silver staining experiments to evaluate the differences in HV, HB, HC and HCC salivary proteins (see FIG. 3A). Silver staining results show that the bands of four groups of salivary proteins are basically consistent, and the number of high-peak protein bands between 10 and 100KDa is basically consistent with the coloring depth (such as wider bands near 70KDa and 60 KDa).
As a result of a Lectin Blotting experiment (Lectin Blotting) performed by DSA (as shown in FIG. 3B), it was observed that the Lectin DSA specifically binds to glycoproteins in saliva samples of four groups, i.e., HV, HB, HC and HCC, and shows about 7 distinct bands, and 1 distinct band (at about 60 kDa) clearly shows in addition to 3 bands having similar binding levels, and it shows that β -D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structures are specifically expressed in saliva of HC patients, and 1 band having a molecular weight of about 35kDa shows β -D-GlcNAc, and Gal β 1-4GlcNAc and (GlcNAc β 1-4) n sugar chain structures are specifically expressed in HV saliva, and that the binding of DSA to HV group is significantly stronger than that of three groups, i.e., HB, HC and HCC. This result substantially coincides with the results of the lectin chip and the saliva chip, and it was confirmed that the expression level of the saliva protein in the mixed sample in which the individual difference was eliminated was limited by the silver-stained chromogenic sensitivity and did not show a significant difference, while the sugar chain structures of β -D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) n recognized by DSA were increased in the saliva glycoprotein of HC patients on the basis of no change in the protein level.
4.3 MALDI-TOF-MS analysis of glycoprotein N-sugar chain separated by DSA-LMPCs
Specific glycoprotein identified by DSA in saliva of HV, HB, HC and HCC patients is separated by LMPCs, then N-sugar chain on the specific glycoprotein is specifically separated by PNGase F endonuclease, mass-to-charge ratio (m/z.) of each sugar chain is identified by MALDI-TOF-MS mass spectrum, each N-sugar chain peak with signal-to-noise ratio S/N >3 obtained by mass spectrum identification is taken and searched for sugar chain database Glycome DB for analysis, N-sugar chain structure information is speculated to be obtained, and mass spectrum data corresponding to each N-sugar chain peak, including m/z., N-sugar chain structure, relative peak intensity and the like, are obtained.
48N-sugar chains were identified in total by MALDI-TOF-MS, of which 20, 13, 26 and 25N-sugar chains were identified in each of HV, HB, HC and HCC groups, respectively. It is noted that the following 11N-sugar chains were present only in the saliva samples of the HC group.
m/z.1996.7238(Fuc)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2006.7316(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2037.7503(Fuc)1(GalNAc)2(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2281.8321(Sia)1(Fuc)1(GalNAc)1(Gal)2(GlcNAc)2+(Man)3(GlcNAc)2；
m/z.2573.9480(Sia)1(Fuc)2(Gal)2(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2772.9572(Sia)2(Gal)4(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2794.0427(Fuc)3(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2(Fuc)1；
m/z.2806.9879(Fuc)1(Gal)6(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2924.0805(Gal)4(GlcNAc)6+(Man)3(GlcNAc)2(Fuc)1；
m/z.3232.1912(Fuc)2(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2；
m/z.3813.3617(Sia)3(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2。
Therefore, it can be finally found that: the saliva sample is detected, and only by determining whether the saliva sample contains any one or any combination of the 11 rock N-sugar chains, the results of liver cirrhosis (HC) screening, early diagnosis, risk assessment, drug screening and/or curative effect assessment can be made.
Claims (6)
1. A product for screening, early diagnosis, risk assessment, drug screening and/or efficacy assessment of liver disease in a saliva sample, comprising:
A. means for obtaining expression levels of three specific glycoprotein sugar chain structures, which are β -D-GlcNAc, Gal β 1-4GlcNAc, and (GlcNAc β 1-4) n, respectively;
B. a marker, a module or a processor for discriminating whether or not the sugar chain structure of the above three specific glycoproteins is highly expressed.
2. The product of claim 1, wherein: the device comprises a lectin chip, an incubation box and a biochip scanning system, wherein at least a lectin DSA is arranged on the lectin chip.
3. The product of claim 1, wherein: the identifier, module or processor is pre-recorded with a reference value corresponding to a Healthy Volunteer (HV) for determining whether high expression is present in comparison to the sample results.
4. Use of a recognition unit for a specific sugar chain for the construction of a product for liver cirrhosis (HC) screening, early diagnosis, risk assessment, drug screening and/or efficacy assessment based on a saliva sample, characterized in that: the recognition unit recognizes any one or any combination of the following 11N-sugar chains:
m/z.1996.7238(Fuc)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2006.7316(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2037.7503(Fuc)1(GalNAc)2(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2281.8321(Sia)1(Fuc)1(GalNAc)1(Gal)2(GlcNAc)2+(Man)3(GlcNAc)2；
m/z.2573.9480(Sia)1(Fuc)2(Gal)2(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2772.9572(Sia)2(Gal)4(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2794.0427(Fuc)3(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2(Fuc)1；
m/z.2806.9879(Fuc)1(Gal)6(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2924.0805(Gal)4(GlcNAc)6+(Man)3(GlcNAc)2(Fuc)1；
m/z.3232.1912(Fuc)2(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2；
m/z.3813.3617(Sia)3(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2。
5. a product for cirrhosis (HC) screening, early diagnosis, risk assessment, drug screening and/or efficacy assessment, on a saliva sample, comprising:
A. lectin DSA;
B. an affinity chromatography solid phase support for coupling lectin DSA;
C. a reagent and a device for separating N-sugar chains from glycoproteins having a β -D-GlcNAc, Gal β 1-4GlcNAc and (GlcNAc β 1-4) N sugar chain structure;
D. a mass spectrometer for displaying m/z values corresponding to the respective N-sugar chains;
E. a label, module or processor for identifying the following 11 rock N-sugar chains from the mass spectrogram to derive a determination of whether any one or any combination of the following 11N-sugar chains are present:
m/z.1996.7238(Fuc)1(GalNAc)1(Gal)1(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2006.7316(Gal)3(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2037.7503(Fuc)1(GalNAc)2(GlcNAc)2+(Man)3(GlcNAc)2(Fuc)1；
m/z.2281.8321(Sia)1(Fuc)1(GalNAc)1(Gal)2(GlcNAc)2+(Man)3(GlcNAc)2；
m/z.2573.9480(Sia)1(Fuc)2(Gal)2(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2772.9572(Sia)2(Gal)4(GlcNAc)3+(Man)3(GlcNAc)2；
m/z.2794.0427(Fuc)3(Gal)3(GlcNAc)4+(Man)3(GlcNAc)2(Fuc)1；
m/z.2806.9879(Fuc)1(Gal)6(GlcNAc)3+(Man)3(GlcNAc)2(Fuc)1；
m/z.2924.0805(Gal)4(GlcNAc)6+(Man)3(GlcNAc)2(Fuc)1；
m/z.3232.1912(Fuc)2(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2；
m/z.3813.3617(Sia)3(Gal)5(GlcNAc)6+(Man)3(GlcNAc)2。
6. the product of claim 5, wherein: the affinity chromatography solid phase carrier for coupling lectin DSA is Fe3O4Magnetic microparticles, sepharose, sephadex or glass microspheres.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004002495A1 (en) * | 2002-06-28 | 2004-01-08 | Glykos Finland Oy | Therapeutic compositions for use in prophylaxis or treatment of diarrheas |
| CN102625915A (en) * | 2009-07-14 | 2012-08-01 | 独立行政法人产业技术综合研究所 | Method for measurement of glycoprotein, method for detection of hepatic diseases, reagent for quantification of glycoprotein, and sugar chain marker glycoprotein as measure of disease conditions of hepatic diseases |
| CN103901212A (en) * | 2014-03-28 | 2014-07-02 | 西北大学 | Glycan microarray for identifying serial liver diseases based on saliva glycan-binding proteins, and application of glycan microarray |
| CN105675893A (en) * | 2016-03-04 | 2016-06-15 | 西北大学 | Lectin chip for detecting carbohydrate chain markers based on blood serum and commonly based on protein in blood serum and saliva as well as kit and application of lectin chip |
| CN105785029A (en) * | 2016-03-04 | 2016-07-20 | 西北大学 | Lectin microarray for detecting carbohydrate chain marker based on protein in saliva, lectin microarray for detecting carbohydrate chain marker jointly based on protein in saliva and serum, kit and applications of lectin microarrays |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2524233A4 (en) * | 2010-01-15 | 2013-07-31 | Univ California | Compositions and methods for detecting cancer |
-
2018
- 2018-09-07 CN CN201811044909.1A patent/CN109212227B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004002495A1 (en) * | 2002-06-28 | 2004-01-08 | Glykos Finland Oy | Therapeutic compositions for use in prophylaxis or treatment of diarrheas |
| CN102625915A (en) * | 2009-07-14 | 2012-08-01 | 独立行政法人产业技术综合研究所 | Method for measurement of glycoprotein, method for detection of hepatic diseases, reagent for quantification of glycoprotein, and sugar chain marker glycoprotein as measure of disease conditions of hepatic diseases |
| CN103901212A (en) * | 2014-03-28 | 2014-07-02 | 西北大学 | Glycan microarray for identifying serial liver diseases based on saliva glycan-binding proteins, and application of glycan microarray |
| CN105675893A (en) * | 2016-03-04 | 2016-06-15 | 西北大学 | Lectin chip for detecting carbohydrate chain markers based on blood serum and commonly based on protein in blood serum and saliva as well as kit and application of lectin chip |
| CN105785029A (en) * | 2016-03-04 | 2016-07-20 | 西北大学 | Lectin microarray for detecting carbohydrate chain marker based on protein in saliva, lectin microarray for detecting carbohydrate chain marker jointly based on protein in saliva and serum, kit and applications of lectin microarrays |
Non-Patent Citations (1)
| Title |
|---|
| 健康人和肝癌患者唾液糖蛋白糖链谱的研究;朱珉之;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20110815(第08期);正文第34、39-40、45-46页 * |
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