Product for breast-cancer-II screening and evaluation based on saliva specific glycoprotein carbohydrate chain structure and application thereof

[0054] Preliminary studies have found that there is a significant difference between the carbohydrate types of breast cancer stage II sialyloglycoprotein and healthy volunteers. The sugar chains recognized by PTL-II are significantly different between HV&BB&BC-I and BC-II ( figure 1 ). Based on this, we designed and completed the following experiments.

[0055] The following describes in detail the relevant verification experiments and analysis of this application, and the specific research and development process of the inventor is not limited to this.

[0056] 1. Research method:

[0057] A lectin PTL-II coupled to the surface of ferroferric oxide nanomagnetic particles is used to make a lectin magnetic particle complex to enrich the specific glycoproteins in each group of saliva mixed samples, and use the PNGase F enzyme digestion method to separate specific sugars The N-sugar chains on the protein are identified by the Matrix-Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrum (MALDI-TOF-MS) and the sugar chain structure is inferred. , Obtained lectin PTL-II identification of healthy female volunteers, benign breast tumor/cyst patients, breast cancer stage I patients, and breast cancer stage II patients with saliva specific glycoprotein sugar chain profiles and compared and analyzed the differences, as follows.

[0058] 1.1. Collection and pretreatment of saliva samples

[0059] The saliva samples of healthy volunteers and breast cancer stage II patients used in this experiment have been strictly approved by Northwest University and the Second Affiliated Hospital of Xi'an Jiaotong University (Human Research Ethics Committees (HRECs)). All volunteers who donated saliva samples and the clinicians who assisted in sampling and guidance were informed, agreed and highly cooperative with the work of this study, and completed the collection of saliva samples under uniform sampling requirements. The specific requirements are: the sample donor must be free of diabetes, and the organs other than the stomach and breast should be free of chronic diseases such as inflammation and tumors. At the time of sampling, the donor must make sure that he has not eaten within 3 hours before collecting saliva and has not eaten within 24 hours. Take the medicine, then rinse the mouth with clean and sterile saline (0.9% NaCl) three times to ensure that the donor’s oral hygiene and no food residues, press the tip of the donor’s tongue against the upper palate and collect the naturally secreted saliva sample under the tongue. To a 2mL centrifuge tube, immediately add 10μL of protease inhibitor (Protease Inhibitor Cocktail, Sigma-Aldrich, USA) in an ice bath for temporary storage. A total of 259 saliva samples were collected under the guidance of clinicians: healthy female volunteers (HV=66), patients with benign breast tumors/cysts (BB=65), patients with breast cancer stage I (BC-I=66), breast cancer II Stage patients (BC-II=62). The specific sample statistics are shown in Table 1.

[0060] Within 12 hours of saliva collection, divide 1mL of saliva into centrifuge tubes. If the amount is less than 1mL, add 1×PBS to make up to 1mL, centrifuge at 10,000g×15min, carefully aspirate the supernatant, and determine the amount of nucleic acid protein. After measuring the concentration by Nano-drop, the protease inhibitor was added in the amount of 1 mg of saliva protein and 10 μL of protease inhibitor, and then 50 μg of each saliva sample was taken and mixed according to the mass. The saliva samples of healthy female volunteers, benign breast tumor/cyst patients, and breast cancer stage I group (HV&BB&BC-I), and breast cancer stage II group (BC-II) were obtained. The concentration of the mixed sample was determined using the BCA protein quantification kit (Biyuntian Biotechnology, Shanghai, China).

[0061] Table 1 Saliva sample information used for breast cancer stage II diagnosis

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[0063]

[0064] 1.2. Preparation of lectin PTL-II magnetic particle complex

[0065] Take 2mg of epoxidized Fe 3 O 4 Put the magnetic particles into a 2mL centrifuge tube, add 1mL of absolute ethanol, repeat the inversion for 2min, place it on a magnetic separator so that the magnetic particles are fully absorbed on the bottom and sides of the centrifuge tube, pour out the ethanol, repeat this 5 times to fully clean the magnetic particles. Wash the cleaned magnetic particles with 1mL coupling buffer, repeat 3 times, add 600μL 0.5mg/mL lectin PTL-II solution (PTL-II dissolved in coupling buffer), fix on a shaker and shake at room temperature React for 6h to fully couple. After coupling, place the centrifuge tube on the magnetic separation rack for separation, remove the liquid, remove the centrifuge tube and add 1mL coupling buffer to wash the PTL-II-magnetic particle complex, invert it repeatedly to resuspend it, and place it in full suspension. Separate on a magnetic separation rack, remove the coupling buffer, and repeat washing 3 times to fully remove the unbound lectin PTL-II to obtain the lectin PTL-II magnetic particle complex.

[0066] 1.3. Separation of glycoprotein recognized by lectin PTL-II

[0067] First, use the binding buffer to wash the lectin magnetic particle complex 3 times, 3 min each time. Make up the volume of saliva protein to 500μL with binding buffer, mix it and add it to the lectin magnetic particle complex, mix it upside down, and incubate it in a shaker at 25°C for 3h. After the incubation is completed, the magnetic field is separated, the supernatant is discarded, and the magnetic particles are washed 5 times with a washing buffer for 3 minutes each. After the cleaning is completed, the magnetic field is separated, the supernatant is discarded, 400 μL of cleaning buffer is added to the magnetic particles, and the magnetic particles are mixed upside down and placed in a shaker, and eluted by shaking at 25°C for 1 hour. Magnetic field separation, collect the supernatant, repeat the previous step once, mix the two collected supernatants, and measure the eluted protein concentration by BCA.

[0068] 1.4. Separation of N-sugar chains

[0069] (1) Take 400μg of glycoprotein separated by PTL-II-magnetic complex and add 8 times volume of 9M Urea/1MNH 4 HCO 3 After mixing the solution, shake at 37℃ for 1h to denature glycoprotein;

[0070] (2) Add 5% volume of 100mM DTT solution mother liquor to the protein solution to make the final concentration of DTT 5mM. After mixing, let it stand at room temperature for 1 hour to reduce the disulfide bonds in the protein;

[0071] (3) Cool the protein solution to room temperature first, add 5% volume of 200mM IAM solution mother liquor to the protein solution to make the final concentration of IAM 10mM, and react for 30min at room temperature and shaking in the dark;

[0072] (4) Transfer the protein solution into a 10K ultrafiltration centrifuge tube, centrifuge at 10000g for 15 minutes at room temperature, discard the filtrate, and add 400μL 40mM NH to the tube 4 HCO 3 Mix the solution by pipetting and repeat the previous step 5 times. Make up the volume to 500μL, add 3μL PNGaseF glycosidase, mix well and incubate overnight in a shaker at 37℃ to digest the N-glycans on glycoproteins;

[0073] (5) Centrifuge at 10000g for 10 minutes, and collect the filtrate. Then add 200 μL of ultrapure water to the tube, mix and centrifuge again to collect the filtrate, mix the two filtrates, and dry with a freeze dryer.

[0074] 1.5. Desalination and purification of sugar chains

[0075] (1) Pre-cleaning: Take out the Hypercarb SPE column (50mg), add 3mL 1M NaOH solution, 3mL ultrapure water, 3mL 30% acetic acid, 3mL ultrapure water to the column in sequence;

[0076] (2) Equilibrium: Add 3 mL 50% ACN/0.1% TFA, 3 mL 5% ACN/0.1% TFA solution to the column in sequence;

[0077] (3) Sample loading: 500 μL of 0.1% TFA was added to the lyophilized N-glycan, and vortexed. Add the dissolved N-glycan to the column, collect the filtrate, load the sample again, and repeat the sample 3 times;

[0078] (4) Cleaning: Add 3mL ultrapure water, 3mL 5% ACN/0.1% TFA to the column in sequence;

[0079] (5) Elution: Add 400 μL of 50% ACN/0.1% TFA to the column, collect the filtrate, repeat once, combine the two collected filtrates, and freeze-dry it with a freeze dryer.

[0080] 1.6, N-glycan mass spectrum analysis

[0081] (1) Add 20μL of sugar chain solution to the lyophilized N-glycan, pipette until the sugar chain is completely dissolved, vortex and centrifuge;

[0082] (2) Take 2μL of the dissolved sugar chain, use a pipette to spot on the MTP Anchorchip 384-point target plate, vacuum dry, and then take 2μL to the original position of the target plate and vacuum dry;

[0083] (3) Take 1μL of 20mg/ml DHB matrix solution and spot it on the crystallized sugar chain sample, and wait for it to vacuum dry;

[0084] (5) The target is set on the computer to identify polysaccharides in the reflection positive ion mode.

[0085] 1.7 Analysis of mass spectrometry data

[0086] Use flexAnalysis software to open the original mass spectrum data, select the mass spectrum peaks in the range of m/z 1000-4000 with a signal-to-noise ratio (SNR) greater than 6, and transfer the selected mass spectrum data to Glycowork beach software. The analysis criteria are: select the molecular weight of the precursor ion , Charge state, precursor ion tolerance is 1, fragment ion tolerance is 0.5, no chemical derivatization, no reducing end modification. Plot secondary fragments and primary mass spectrum results.

[0087] The calculation method of the relative abundance of a single sugar chain: add the signal values ​​of all sugar chains as the denominator (N), divide the signal value (n) of a single sugar chain by N, that is, n/N is the relative abundance of a single sugar chain .

[0088] 2. Research results:

[0089] A total of 87 signal peaks (SNR greater than 6) were identified by MALDI-TOF-MS. There are 54 signal peaks that can be resolved to the sugar chain structure, of which 51 are N- sugar chain peaks identified by PTL-II, as shown in Table 2. Shown. In HV&BB&BC-I and BC-II groups, 29 and 39 N sugar chains recognized by PTL-II were identified, and their relative abundances were 73.21% and 71.94%, respectively.

[0090] Table 2. Sugar chains separated from two sets of samples

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[0096] *Note: blue (dark) square: N-acetylglucosamine; red triangle: fucose; green (dark) circle: mannose; yellow (light) circle: galactose; yellow (light) (Color) square: N-acetylgalactosamine; purple diamond: sialic acid; /: represents that the corresponding sugar chain is not detected in the sample.

[0097] Any one or any combination of 22 kinds of N-sugar chains was only present in the saliva samples of the BC-II group. The details are as follows (relative abundance in parentheses):

[0098] (1)m/z 1521.035

[0099] (2)m/z 1721.556

[0100] (3)m/z 1770.642/1792.624

[0101] (4)m/z 1850.666

[0102] (5)m/z 1973.721

[0103] (6)m/z 2004.716

[0104] (7)m/z 2018.639

[0105] (8)m/z 2177.821

[0106] (9)m/z 2190.766

[0107] (10)m/z 2193.816

[0108] (11)m/z 2392.851

[0109] (12)m/z 2496.862

[0110] (13)m/z 2507.914

[0111] (14)m/z 2636.957

[0112] (15)m/z 2677.959

[0113] (16)m/z 2703.986

[0114] (17)m/z 2830.999

[0115] (18)m/z 2937.051

[0116] (19)m/z 3054.144

[0117] (20)m/z 3212.126

[0118] (21)m/z 3253.153

[0119] (22)m/z 3471.232

[0120] Therefore, in the end, it can be concluded that the detection of saliva samples only needs to determine whether it contains any or any combination of these 22 N-sugar chains to make breast cancer stage II screening, early diagnosis, risk assessment, Results of drug screening and/or efficacy evaluation.