CN111157661A

CN111157661A - By using TiO2Method for comprehensively analyzing tree shrew respiratory tract tissue sugar chain spectrum by PGC chip mass spectrometry

Info

Publication number: CN111157661A
Application number: CN202010027034.5A
Authority: CN
Inventors: 杨子峰; 王静蓉; 丘利芳; 陈家敏; 杨春光; 叶孙威; 康玥; 麦芷桐; 佟天天
Original assignee: Guangzhou Institute Of Respiratory Health; State Key Laboratory Of Quality Research In Chinese Medicine (macau University Of Science And Technology); First Affiliated Hospital of Guangzhou Medical University
Current assignee: Guangzhou Institute Of Respiratory Health; State Key Laboratory Of Quality Research In Chinese Medicine (macau University Of Science And Technology); First Affiliated Hospital of Guangzhou Medical University
Priority date: 2020-01-10
Filing date: 2020-01-10
Publication date: 2020-05-15

Abstract

The invention discloses a method for utilizing TiO₂-PGC chip mass spectrometry method for comprehensive analysis of sugar chain spectrum of tree shrew respiratory tract tissue, comprising the following steps: collecting the respiratory tract tissues of the tree shrews; cleaving the sugar chains of the tree shrew respiratory tract tissue; by TiO₂-PGC chip-Q-TOF-MS assay for N-sugar chain spectra; identifying sialic acid linkages by sialidase reaction; by TiO₂PGC chip-QQQ-MS method quantitative analysis of N-sugar chain spectrum. The method can accurately and comprehensively realize qualitative and quantitative analysis of the sugar chain spectrum of the tree shrew respiratory tract tissue.

Description

By using TiO2Method for comprehensively analyzing tree shrew respiratory tract tissue sugar chain spectrum by PGC chip mass spectrometry

Technical Field

The invention belongs to the field of biochemical medicine, and particularly relates to a method for preparing a titanium dioxide film by using TiO₂-PGC chip mass spectrometry method for comprehensive analysis of glycosylation on the cell surface of respiratory tract tissue of tree shrew of influenza virus animal model.

Background

Influenza virus infection HAs caused about 4 million cases to occur, of which more than 3 million deaths occur in the united states from 10 months to 2019 years in 2018 Hemagglutinin (HA) -sialylated sugar chains bind to bring the virus into contact with host cells, which is a crucial step for influenza virus infection, generally avian influenza virus preferentially binds to glycans ending with

α

2,3 Sialic Acid (SA), while human influenza virus preferentially recognizes glycans ending with

α

2,6 SA.

The establishment of animal models aiming at simulating the progress of human virus infectious diseases is indispensable to the pathogenesis of the human virus infectious diseases and the prevention of the diseases. Rodent models such as mice, rats, guinea pigs, and hamsters are widely used for the study of influenza virus, but none of them exhibit some human clinical symptoms. Furthermore, ferrets and non-human primates (e.g., macaques and apes) can exhibit a variety of clinical symptoms similar to those of humans. However, their availability, ethics and high cost have limited the use of research. Recent studies found that tree shrews (Tupaia belangeri) had been developed as an alternative animal model for studying influenza virus. Furthermore, such animals are widely distributed in southeast Asia, south China and southwest, are genetically similar to humans, and are considered to be "lower primates". Tree shrew has also been used as an animal model for the study of human influenza virus H1N1, avian influenza virus H9N2 and influenza b. Therefore, they are ideal animal models for the study of viral infectious diseases based on their small size, homology to humans (e.g., anatomy, physiology, immunology and disease progression), and susceptibility to human viruses.

Lectin histochemistry is a standard method for detecting SA ligation and has been widely used to study the expression and distribution of α 2,3SA and α 2,6SA ligation receptors on host cells Maackia amurensis (MAA) lectins (MAA 1and MAA2) were used to detect α 2,3SA and Sambucus Nigra (SNA)

α

2,6 SA. however, lectin experiments cannot quantify and provide all-or-nothing results.

Disclosure of Invention

The invention aims to solve the technical problems and provides a method for accurately and comprehensively analyzing the sugar chain spectrum of the tree shrew respiratory tract tissue.

In order to achieve the above object, the present invention provides the following technical solutions:

by using TiO₂-PGC chip mass spectrometry method for comprehensive analysis of sugar chain spectrum of tree shrew respiratory tract tissue, comprising the following steps:

collecting tree shrew respiratory tract tissues;

secondly, cracking sugar chains of the tree shrew respiratory tract tissues;

step three, passing through TiO₂-PGC chip-Q-TOF-MS assay for N-sugar chain spectra;

step four, identifying the sialic acid connecting bond through sialidase reaction;

step five, passing through TiO₂PGC chip-QQQ-MS method quantitative analysis of N-sugar chain spectrum.

According to a preferred embodiment of the invention, said step two is carried out as follows:

1) cutting tree shrew respiratory tract tissue into fragments, dissolving and cracking the fragments by RIPA, and extracting protein;

2) centrifuging, filtering and concentrating the extracted protein; determining the protein concentration of each sample; each sample was taken in equal mass and diluted to 1 μ g/μ L per 200 μ g protein with 100mM ammonium bicarbonate pH 7.4; then PNGaseF was added, incubated at 37 ℃ for 16 hours, and cleaved to obtain N-sugar chains directly loaded on the pretreated C₁₈Dissolving on the column with distilled water, mixing the flow-through solution and the dissolving solution, freeze drying, re-dissolving the lyophilized sample with 100 μ L distilled water, and centrifuging at 14000rpm at 4 deg.C for 5 min.

According to a preferred embodiment of the present invention, the third step is performed as follows:

separating the neutral N-sugar chains and the acidic N-sugar chains of each sample into two independent sample injections; injecting 2 mu L of sample for the first time, transferring the sample to an enrichment column at the flow rate of 3 mu L/min by loading a buffer solution, wherein the buffer solution consists of 0.6 percent by weight of formic acid, 2 percent by weight of acetic acid and 2 percent by weight of acetonitrile which are dissolved in water; the mobile phase of the nano pump is respectively composed of 1 percent of acetic acid dissolved in water as phase A and acetonitrile as phase B; washing with gradient solution 5% B phase for 6min, 5-60% B phase for 10min, and 80% B phase for 3min, eluting neutral sugar from the analytical column at flow rate of 0.5 μ L/min; injecting 5 mu L of ammonia water for the second sample injection, eluting the mobile phase of the acidic N-sugar chains from the analytical column by taking 0.5 percent acetic acid water solution as an A phase and 1 percent acetic acid-acetonitrile solution as a B phase, and carrying out gradient elution by taking 5 percent B phase for 1min, 5-60 percent B phase for more than 10min and 80 percent B phase for 3min, wherein the balance time before the injection is 10 min;

obtaining N-sugar chain spectrum from Q-TOF MS, dried N₂The sample was treated at 11L/min and 225 ℃ and MS/MS spectra were obtained by mass range m/z 500-3000 and m/z 100-3000 positive mode respectively, mass corrected by reference mass size m/z922.0098 and collision energy set at 10-40 eV.

According to a preferred embodiment of the present invention, said step four is performed as follows:

mu.L of sialidase was mixed with 14. mu.L of the mixed sample and 4. mu.L of 5 Xreaction solution to give 20. mu.L of reaction system, incubated at 37 ℃ for 1 hour, and then subjected to mass spectrometry.

According to a preferred embodiment of the present invention, the step five is performed as follows:

obtaining N-sugar chain spectrum from QQQ MS, and drying N in positive mode by multiple reaction monitoring method₂The sample was treated at 11L/min and 225 ℃ with the residence time of neutral N-glycans set to 10ms and the acid N-sugar chains set to 5ms, the fragmentation voltage set to 380V, and the RF voltage amplitudes of the high and low voltage ion funnels set to 150 and 200V, respectively.

Preferably, the method of the present invention further comprises data processing and data analysis, in which multivariate data analysis is performed on mass spectrum data by computer software, and these mass spectrum data are compared with data of an existing sugar chain database to identify differences and characterization of N-sugar chains expressed on tissues.

The respiratory tract tissue refers to the tissue of a channel through which air flows when the lung breathes, and comprises the tissues of organs such as nose, pharynx, larynx, trachea, bronchus, lung and the like, including but not limited to the respiratory tract tissue from any one or more of turbinates, soft palate, trachea, bronchus and lung.

TiO of the invention₂The PGC chip comprises a 75 μm by 150mm PGC analytical column (PGC, 5 μm) preceded by a Sandwich enrichment column consisting of two 100nL PGC columns (PGC1, PGC2) sandwiching a 45nL TiO column₂And (4) column composition.

The invention can comprehensively analyze the glycosyl receptor on the surface of the respiratory tract tissue cell of the tree shrew of the model of the influenza animal. First by the newly established TiO₂And (3) carrying out qualitative analysis on the sugar chain structure of the surface glycosyl receptor of the tree shrew by a PGC chip-Q-TOF-MS method for the first time, representing the structural diversity of the N-sugar chains of the respiratory tract of the tree shrew, and identifying 219N-sugar chains in total. Furthermore, each N-sugar chain is quantified by a highly sensitive and accurate MRM method using¹³C marked internal standard is used for correcting the inherent difference between runs in mass spectrum detection, and the result shows that the N-sugar chains in the turbinates and lung tissues of the tree shrew have obvious difference with the N-sugar chains in the soft palate, trachea and bronchus. Analyzing the result by the method of the invention to speculate the nose of the tree shrewThe 28N-sugar chains with high abundance levels in the nail bone may be associated with infection with influenza A/California/04/2009(H1N 1). The invention comprehensively analyzes the sugar chain of the respiratory tract of the tree shrew for the first time, and has better understanding on human influenza infection and pathogenesis.

Drawings

FIG. 1 is a chromatogram of extracted compounds (ECC) of N-sugar chains in a tree shrew respiratory tract tissue.

FIG. 2 is a chromatogram of sugar chain 5_4_1_1(A) before and after sialidase S treatment.

Fig. 3 is a mass spectrum of diagnostic debris identifying NeuGc and NeuAc types.

FIG. 4 is a graph of OPLS-DA scores of N-sugar chains in respiratory tract tissues of five tree shrews.

FIG. 5 is a graph of the 6 potential influenza CA04 receptors in the tree shrew turbinates.

FIG. 6 is a graph of another 6 potential influenza CA04 receptors in the tree shrew turbinates.

FIG. 7 is a graph of another 6 potential influenza CA04 receptors in the tree shrew's turbinates.

FIG. 8 is a graph of another 6 potential influenza CA04 receptors in the tree shrew's turbinates.

FIG. 9 is a graph of the additional 4 potential influenza CA04 receptors in the tree shrew's turbinates.

Detailed Description

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative of the present invention only, and are not intended to limit the scope of the present invention.

Unless otherwise specified, the percentages (%) in the present invention are percentages by weight.

Example 1:

TiO₂-PGC chip-Q-TOF-MS method and TiO₂Comprehensive analysis of N-sugar chain spectrum by the-PGC chip-QQQ-MS method

Briefly, chromatographic separation of N-sugar chains by custom-made TiO₂PGC chips (Agilent, Waldbronn, Germany) were carried out on an Agilent 1260 affinity HPLC chip liquid chromatography system (Agilent, Santa Clara, Calif.). The chip has a 75 μm × 150mm PGC analytical column (PGC, 5 μm) andone sandwich enrichment column of Sandwich sample before PGC analytical column-two 100nL PGC columns (PGC1, PGC2) sandwich one 45nLTiO column₂And (4) column composition.

N-sugar chain spectra were obtained from Agilent 6550iFunnel Q-TOF MS. Dried N₂The samples were treated at 11L/min and 225 ℃. MS and MS/MS spectra were obtained by positive mode with mass ranges of m/z 500-3000 and m/z 100-3000, respectively. The mass correction is performed by reference to the mass size m/z 922.0098. The collision energy was set to 10-40 eV.

Sialidase S is an enzyme that cleaves α 2,3-SA, the mode of attachment of the identified N-sugar chain SA is then determined by the sialidase S (sialidase S) reaction.

1. Experimental Material

1.1 materials and chemistry

¹³C-labelled Internal Standards (ISs) comprising¹³C flag 5_4_1_0,¹³C markers 5_4_1_1(A) (sia α 2,3) and¹³c-tag 5_4_1_1(a) (sia α 2,6), a product from Asparia Glycomics (San Sebastian, spain.) PNGase F (500,000 units/mL) from New England BioLabs (Beverly, massachusetts.) glycosyl sialidase S and dyes to determine protein concentration were obtained from Prozyme (Hayward, california) and Bio-Rad (Hercules, california), radioimmunoprecipitation assay buffer (RIPA buffer) was obtained from Thermo (Waltham, massachusetts) bovine serum albumin (milbara), sodium pentobarbiturate, Phosphate Buffer Solution (PBS), ammonium bicarbonate, aqueous ammonia solution, formic acid (AA) and acetic acid (FA) were obtained from Sigma (st. louis, milicone, milona, U.S. and millionta-0.inf, 0.0. county. millite) and were obtained from Millipore r g. filter (r 3, Millipore Q-r, Millipore, r, C-3, C-4-1, a product from asprellics, separian, septoria.

1.2 animals

Healthy adult female tree shrews with the weight of 100-130g were purchased from the animal experiment center of Kunming medical university (Yunnan, China). Animals were kept in cages in the same space, and were allowed to drink and drink water at 20 ± 0.5 ℃ in a 12-hour cycle, the study was in accordance with the regulations of the experimental animal management in Guangdong province (2010). The study protocol was reviewed and approved by the institutional animal Care and use Committee of Kunming science and technology university medical laboratory animals (IACUC: KMU 2017-M0016).

2. Experimental methods

2.1 Collection of respiratory tract tissues of Tree shrews

Six tree shrews were first anesthetized with 3% PBS-dissolved sodium pentobarbital (30mg/kg), and then five respiratory tissues, turbinates, soft palate, trachea, bronchi and lungs, were collected separately and stored at-80 ℃ for further manipulation.

2.2 protein Capture and cleavage of sugar chains from respiratory tissues

Respiratory tract tissues were washed with precooled PBS and then cut to approximately 1mm³And (4) fragmenting. Add 1mLRIPA solution to each 10mg tissue for lysis, incubate on ice for 1h, vortex every 10 min. Centrifuge at 14000rpm for 15min, buffer exchange the supernatant with water and concentrate to about 30 μ L using a 3K centrifuge filter. The protein concentration of each sample was determined by the bradford method using BSA as a standard. Then, 200. mu.g of protein was taken from each sample, and diluted to 1. mu.g/. mu.L with 100mM ammonium bicarbonate (pH 7.4). 2 μ L of PNGase F was then added and incubated for 16h at 37 ℃. Then, the N-sugar chains obtained by cleavage were directly loaded on a pretreated C18 column, and dissolved out with 1mL of distilled water. Mixing the flow-through solution and the dissolving solution, and freeze drying. The lyophilized sample was reconstituted with 100. mu.L of distilled water and centrifuged at 14000rpm at 4 ℃ for 5 min. QC samples were pooled with equal amounts from different tissues for N-glycan analysis.

2.3 adding an internal standard to the sample for quantitative analysis

Three are¹³C-labelled internal standard comprising 13C-labelled 5_4_1_0, 13C-labelled 5_4_1_1(A) (sia α 2,3) and¹³c-labeled 5_4_1_1(A) (sia α 2,6), mixed and diluted with water to 500 nM. and then 20. mu.L of each sample was mixed with 5. mu.L of the mixed 13C-labeled internal standard solution before quantitative analysis.

2.4TiO₂-PGC chipAcquisition of N-sugar chain spectra by-Q-TOF-MS

Sugar chain spectrum of TiO reported from the previous literature₂And obtaining the product by a PGC chip-Q-TOF-MS method. Briefly, chromatographic separation of N-sugar chains by custom-made TiO₂PGC chips (Agilent, Waldbronn, Germany) were carried out on an Agilent 1260 affinity HPLC chip liquid chromatography system (Agilent, Santa Clara, Calif.). TiO 2₂The PGC chip has a 75 μm × 150mm PGC analytical column (PGC, 5 μm) and a Sandwich enrichment column in front of the PGC analytical column, a 45nL TiO column sandwiched by two 100nLPGC columns (PGC1, PGC2)₂And (4) column composition.

The neutral N-sugar chains and acidic N-sugar chains of each sample were split into two separate injections. For the first injection, 2. mu.L of sample was injected and transferred to the enrichment column via loading buffer (0.6% AA, 2% FA, 2% acetonitrile in water) at a flow rate of 3. mu.L/min. The mobile phase of the nano pump consists of 1% FA dissolved in water (A) and acetonitrile (B) respectively. By gradient solution: washing with 5% B for 6min, 5-60% B for 10min, and 80% B for 3min, and eluting neutral sugar from the analytical column at flow rate of 0.5 μ L/min. And injecting 5 mu L of ammonia water for the second injection. The mobile phase for eluting the acidic N-sugar chains from the analytical column was composed of a 0.5% aqueous solution of FA (adjusted to pH3.0 with aqueous ammonia) (A) and a 1% solution of FA-acetonitrile (B). Gradient eluting with 5% B for 1min, 5-60% B for more than 10min, and 80% B for 3 min. The equilibration time before needle absence was 10 min.

2.5 identification of sialic acid linkages by sialidase reaction

mu.L of sialidase (sialidase S) was mixed with 14. mu.L of 5 kinds of tree shrew respiratory tract tissue sugar chain mixed samples (as described in 2.2) and 4. mu.L of 5 Xreaction solution (250nM sodium phosphate pH6.0) to prepare a 20. mu.L reaction system, which was incubated at 37 ℃ for 1 hour and then subjected to mass spectrometry.

2.6TiO₂PGC chip-Quantitative analysis of N-sugar chain by QQQ-MS method

TiO₂The PGC chip-Q-TOF-MS method was used for qualitative analysis of N-sugar chains. Chromatographic conditions with TiO₂PGC chip-Q-TOF-MS analysis was identical. N-sugar chain quantification was performed by Agilent 6490iFunnel QQQ MS, in positive mode using Multiple Reaction Monitoring (MRM). Dried N₂The samples were treated at 11L/min and 225 ℃. The retention time of the neutral N-glycans was set to 10ms, while that of the acidic N-sugar chains was set to 5 ms. The fragmentation voltage was set at 380V, while the RF voltage amplitude of the high and low voltage ion funnels was 150 and 200V, respectively.

2.7 data processing and data analysis

All MS and MS/MS data were done by Agilent MassHunter Qualitative Analysis B.06.00 software. N-sugar chains were mined in different tissues by the formula algorithm (FBF). Establishing a matching standard: personal N-carbohydrate chain databases were imported as a source of formulas, each allowing a minimum of 10 possible matches. Allowing the adduct matching with protons (+ H) and sodium (+ Na) with charge range of 1-3 in positive mode; the mass tolerance is set to be +/-10 ppm; all scores (including quality scores, isotopic abundance scores, isotopic distance scores) < 60 were filtered as results.

All MRM (mass spectrometry multiple reaction monitoring) data were done by MassHunter Quantitative analysis b.06.00 software. Each N-sugar chain was quantified as the ratio of its peak area to the corresponding internal standard.¹³C label 5_4_1_0 as an internal standard for calibration of neutral sugars, and internal

standards sia α

2,3 and

sia α

2,6 for acidic sugars:¹³c flag 5_4_1_1(A) and¹³c flag 5_4_1_1(a) (sia α 2, 6).

Multivariate data analysis was performed by version SIMCA15.0.2 (Sartorious Stedim Biotech, Umea, Sweden). Differences in N-sugar chains expressed on different tissues were identified by the orthogonal partial least squares discriminant analysis (OPLS-DA) based on the variable importance map (VIP) values. Data differencing was performed by GraphPad Prism 6(GraphPad software, La Jolla, CA, USA) and differences were considered to be between the two sides with a P value < 0.05.

3. Results of the experiment

3.1 comprehensive characterization of N-sugar chains in respiratory tract tissue of Tree shrew

TiO₂The structural diversity of tree shrew respiratory tract N-carbohydrate chains is disclosed for the first time by a PGC chip-Q-TOF-MS method. Based on the high resolution MS and MS/MS data, a total of 219N-sugar chains were identified from the QC samples, containing 28 neutral sugars (from 17 components) and 191 acidic sugars (from 88 components). An Extracted Compound Chromatogram (ECC) in which N-sugar chains were detected in the flow-through phase (neutral sugars) and the eluate (acidic sugars) is shown in FIG. 1.

The SA in the sugar chain can be linked to galactose through α,3 or α,6 two glycosidic linkages, of which α,3-SA and α,6 linked SA are considered as receptors for avian and human influenza virus, respectively.thus, analysis of the way of SA linkage on the N-sugar chains of the respiratory tract of tree shrew can deepen the understanding of the mechanism of infecting tree shrew with influenza virus.in this study, the way of linkage of the identified N-sugar chain SA was determined using the sialidase S reaction.sialidase S is an enzyme that cleaves α 2, 3-SA.5 _4_1_1(A) produced 5 isotopes as shown in FIG. 2, the peaks remained unchanged at 8.87 and 9.15min, whereas after treatment with sialidase S, the peaks disappeared at 9.30, 9.62 and 9.87min, indicating that the two isotopes eluted at 8.87 and 9.15min were α,6-SA, while the peaks eluted at 9.30, 9.62 and 9.87min were found to be more than three isotopes, namely, α,6-SA, 3-9.9.9.9.9.9.9, 3-9.9.87 min, and 3.9.9.9.9.9.9.9.9.9.9 min.

In addition, among the identified acidic sugars, there are two types of SA, NeuAc and NeuGc, respectively. Both types of SA have the same structure except for an additional oxygen atom in the N-glycosyl group of NeuGc. This subtle difference leads to the creation of N-sugar chain structures from two species, multiple isotopes, making identification challenging. However, these two types of SAs are distinguished with the help of MS/MS data acquired from Q-TOF-MS. Fragment ions 274.1(NeuAc-H2O), m/z 292.1(NeuAc) and m/z 657.2(Hex1HexNac1NeuAc1) were used as N-glycans diagnostic fragments containing NeuAc, while fragment ions m/z 290.1(NeuGc-H2O), m/z 308.1(NeuGc) and m/z673.2(Hex1HexNac1NeuGc1) were used as N-glycans diagnostic fragments containing NeuGc. For example, as shown in FIG. 3A, the formula is C71H118N4O54 at the peak of 9.26min, based on which the [ M +2H ]2+ ion is M/z 946.3349. Two N-sugar chains were matched to this molecular formula in our personal database, including 6_3_0_1(A) and 5_3_1_1 (G). Again, the diagnostic fragment of NeuAc (m/z 292.1and m/z 657.1) confirmed from MS/MS that this peak was 6_3_0_1 (A). In the same manner, of the 191 acidic sugars identified from the tree shrew respiratory tract, 151 (from 60 components) were identified as NeuAc type and 37 (from 26 components) were identified as NeuGc type. It is noted that there are 3N-sugar chains of mixed type, and NeuAc and NeuGc, including 6_4_0_2(1A1G) -a,6_4_0_2(1A1G) -B (fig. 3B) and 5_5_0_2(1 A1G).

Both NeuAc and NeuGc can act as influenza virus hemagglutinin protein (HAs) receptors, and NeuGc specificity is a species-specific behavior. Therefore, it is important to identify NeuGc and NeuAc on the cell surface of respiratory tract. The respiratory tract of pigs and mice is rich in NeuGc, but not in humans and ferrets. This is because NeuGc expression is associated with cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), which hydroxylates NeuAc to NeuGc. It is noteworthy that the study of the present invention reported for the first time that tree shrew respiratory N-sugar chains had both NeuAc and NeuGc types. Furthermore, the CMAH gene in the tree shrew provides a theoretical basis for the NeuGc in the tree shrew.

3.2N-sugar chain composition of different respiratory tract tissues of tree shrew

The identified N-sugar chains are then passed through TiO₂The MRM pattern of the PGC chip-QQQ-MS was quantitatively analyzed. The product ion and fragmentation energy were optimized for each N-sugar chain to achieve the highest signal. The MRM transition was used to monitor each of the N-sugar chains listed in Table S1. According to the levels of each N-sugar chain of five respiratory tract tissues of the tree shrew obtained by analysis, the mannose-rich form in neutral sugar is the most dominant, wherein the abundance is the highest in 9_2_0_ 0. Furthermore, the abundance of acidic N-sugar chains in each respiratory tract tissue is much higher than that of neutral sugars, with the acidic N-sugar chains containing one fucose being the most abundant. Furthermore, the overall level of N-sugar chains in the lungs is higher than in other respiratory tracts.

The MRM method exhibits high sensitivity due to its two selective steps of eliminating background ionsSensitivity. This method simultaneously exhibits high dynamic range up to 5 orders of magnitude. And each N-sugar chain was quantified as the ratio of its corresponding internal standard. The internal standard is used for correcting the inherent inter-operation difference in MS and simultaneously carrying out more reasonable comparison on the N-sugar chain components in different respiratory tract tissues of the tree shrew. By three¹³C-labeled N-sugar chains as internal standards, two for each N-acetylglucosamine (GlcNAc) of each internal standard¹³The C atom is labeled, and the mass shift of the C atom is 8Da compared with other natural sugar chains. These labeled internal standards have the same chromatographic retention time and ionization reaction advantages as the target N-sugar chains in the sample.

Multivariate analysis was then performed to analyze the different respiratory tract tissues of the tree shrews. As shown in fig. 4, the OPLS-DA score plots the soft palate, trachea and bronchi together, while the turbinates and lungs do not overlap with other parts of the respiratory tract (R2X-0.583, R2Y-0.873, Q2-0.823). The above results show that the N-sugar chain composition of the turbinates and lungs of tree shrews are significantly different from those of the soft palate, trachea and bronchi. Furthermore, N-sugar chains with a VIP value > 1 are important variables for the OPLS-DA model. Thus, 112 potential N-sugar chain markers were found to be useful for distinguishing between different respiratory tract tissues.

Example 2:

1. experimental Material

1.1 viruses and cells

H1N1 virus A/California/04/2009(CA04) and H9N2 virus A/Duck/Hong Kong/Y280/97(Y280) were given gifts by professor J.S.M.peiris (Special administrative area of Hong Kong, Hong Kong Hoccdigital college of Lijiacheng medical college, Hong Kong, China). Madin-Darby canine kidney cells (MDCK) were purchased from the american biological standards resource center (ATCC) (Manassas, VA, USA) and cultured in DMEM medium (Gibco, New Zealand) plus 10% Fetal Bovine Serum (FBS) (Gibco) at 37 ℃, under 5% CO2 environment. H1N1 virus was inoculated in DMEM-cultured MDCK cells containing 1. mu.g/mL of TPCK-treated pancreatin (Sigma) and diabody (Gibco). All experiments were performed in a biosafety level two laboratory.

1.2 animals

Healthy adult female tree shrews with the weight of 100-130g were purchased from the animal experiment center of Kunming medical university (Yunnan, China). Animals were kept in cages in the same space, and were allowed to drink water freely in a 12-hour cycle, with the temperature being maintained at 20. + -. 0.5 ℃. The study was in compliance with the regulations of the Experimental animals administration in Guangdong province (2010). The study protocol was reviewed and approved by the institutional animal Care and use Committee of Kunming science and technology university medical laboratory animals (IACUC: KMU 2017-M0016).

1.3 materials and chemistry

¹³C-labelled Internal Standards (ISs) comprising¹³C-labeled 5_4_1_0、¹³C-labeled 5-4-1-1 (A) (sia α 2,3) and¹³c-labelled 5-4-1-1 (A) (sia α 2,6), a product from Asparaa Glycomics (San Sebasitan, Spain.) PNGase F (500,000 units/mL) was obtained from New England Biolabs (Beverly, Mass.) glycosyl sialidase S and dyes to determine protein concentration were obtained from Prozyme (Hayward, Mass.) and Bio-Rad (Hercules, California, U.S.A.) radioimmunoprecipitation assay buffer (RIPA buffer) was obtained from Thermo (Waham, Mass.) bovine serum albumin, sodium pentobarbital, Phosphate Buffer Solution (PBS), ammonium bicarbonate, ammonia solution, formic acid (AA) and acetic acid (FA) were obtained from Sigma, Louis, Miliace, Millipore 0.7, Septori K-0.84, and Waterson acetonitrile (Centra-filter, America) were obtained from Watersolar (Avalon Job & Addison & E.

2. Experimental protocol

2.1 Tree shrew Virus inoculation and Virus titer measurement

Three tree shrews determined to be serologically negative were selected for this experiment. Animals were first anesthetized and then inoculated nasally with about 105TCID 50A/California/04/2009 (H1N 1). Turbinates, trachea, lungs were collected 2 days after infection. After the tissue was weighed, 10mL/g of MEM medium containing 1. mu.g/g of pancreatin treated with mLTPCK and a diabody was added thereto, and the mixture was homogenized. The supernatant was collected and the virus titer was determined in MDCK cells by the method of TCID 50.

2.2TiO₂Quantitative analysis of N-sugar chain by the-PGC chip-QQQ-MS method

N-sugar chain quantification was performed by Agilent 6490iFunnel QQQ MS, in positive mode using Multiple Reaction Monitoring (MRM). Dried N₂The samples were treated at 11L/min and 225 ℃. The retention time of the neutral N-glycans was set to 10ms, while that of the acidic N-sugar chains was set to 5 ms. The fragmentation voltage was set at 380V, while the RF voltage amplitude of the high and low voltage ion funnels was 150 and 200V, respectively.

2.3 data processing and data analysis

3. Results of the experiment

3.1N-sugar chains associated with influenza Virus infection

In order to reveal the N-sugar chains associated with influenza virus infection, tree shrew was inoculated with CA04 influenza virus in this study. As a result, CA04 was recovered in all three turbinate samples, while CA04 was not recovered in the lung sample (table 1). The above results indicate that the tree shrew's turbinates are more susceptible to CA04 than the lungs and trachea. Since recognition of influenza virus HA with sugar structures on the host cell surface is a crucial step in virus entry and infection, the abundant N-sugar chains in the turbinate are considered as potential receptors for CA 04. Generally, the overall level of N-sugar chains in the lung is much higher than in other respiratory tissues. However, the content of 28 sugar chains in the turbinates was significantly higher than that in the lungs (fig. 5 to 9). Therefore, these N-sugar chains are presumed to be potential acceptors of CA 04. Previous studies reported that the turbinates, trachea and lungs were not susceptible to A/Duck/HongKong/Y208/97(H9N2) virus (Table 1). This suggests that these 28N-sugar chains may be specific for CA 04. But further experiments in the future are required to demonstrate.

TABLE 1 sensitivity of different respiratory tissues (turbinate, trachea and lungs) to H1N1 virus A/California/04/2009 and H9N2 virus A/Duck/HongKong/Y208/97.

^aIntranasal inoculation of Tree shrews to about 10⁵TCID 50H 1N1 Virus A/California 04/2009. Samples were collected from 3 tree shrews 2 days after inoculation.

^bTissue tropism of Tree shrews infected with H9N2 Virus A/Duck/HongKong/Y208/97 2 days after inoculation (data from previous studies)

Claims

1. By using TiO₂-PGC chip mass spectrometry method for comprehensively analyzing the sugar chain spectrum of tree shrew respiratory tract tissue, which is characterized by comprising the following steps:

collecting tree shrew respiratory tract tissues;

secondly, cracking sugar chains of the tree shrew respiratory tract tissues;

2. The method of claim 1, wherein step two is performed as follows:

2) centrifuging, filtering and concentrating the extracted protein; determining the protein concentration of each sample; each sample was taken in equal mass and diluted to 1 μ g/μ L per 200 μ g protein with 100mM ammonium bicarbonate pH 7.4; then PNGase F was added, incubated at 37 ℃ for 16 hours, and cleaved to obtain N-sugar chains directly loaded on the pretreated C₁₈Separating with distilled water, mixing the two flowsAnd (4) introducing the solution and the dissolved solution, freeze-drying, re-dissolving the freeze-dried sample with 100 mu L of distilled water, and centrifuging for 5min at 14000rpm under the environment of 4 ℃.

3. The method of claim 1, wherein step three is performed as follows:

4. The method of claim 1, wherein step four is performed as follows:

5. The method of claim 1, wherein step five is performed as follows:

6. The method according to claim 1, further comprising data processing and data analysis, wherein the difference and characterization of the N-sugar chains expressed on the tissue is identified by performing multivariate data analysis on mass spectrum data by computer software and comparing the mass spectrum data with data of an existing sugar chain database.

7. The method of claim 1, wherein the airway tissue comprises tissue of a channel through which airflow passes as the lungs breathe.

8. The method of claim 1, wherein the TiO is selected from the group consisting of₂The PGC chip comprises a 75 μm × 150mm PGC analysis column, a Sandwich enrichment column is arranged in front of the PGC analysis column, and the sandwich of two 100nL PGC columns and 45nL TiO is arranged in the sandwich₂And (4) column composition.