Plasma protein enrichment and mass spectrometry identification based on magnetic helical multi-walled carbon nanotubes modified with benzophenonesulfonic acid

Abstract

This article provides a method for selectively enriching low-abundance proteins from a sample containing multiple proteins, comprising the steps of: 1) adding carbon nanotubes modified with pyrenesulfonic acid to the sample so that a portion of the multiple proteins are adsorbed to the surface of the carbon nanotubes; and 2) separating the carbon nanotubes from the sample. This article also provides a method for identifying the types and contents of proteins in a sample enriched with low-abundance proteins by mass spectrometry. The method provided herein uses C12Im‑Cl to remove non-specifically adsorbed proteins, combined with a high-resolution mass spectrometry data acquisition method, which can achieve the qualitative and quantitative analysis of more than 2,000 plasma proteins in a single mass spectrometry injection.

Description

Technical Field

[0001] The present invention relates to a method for selectively enriching low-abundance proteins, in particular to a method for enriching low-abundance proteins from plasma samples, and also relates to a method for identifying the types and contents of proteins in a sample enriched with low-abundance proteins by mass spectrometry. Background Art

[0002] Plasma is the most commonly used clinical sample. Compared with other body fluids (such as cerebrospinal fluid, gastric juice, bile and synovial fluid), plasma is easy to obtain, and sampling and storage are easier to standardize. Changes in proteins in plasma can indicate the occurrence and progression of a variety of diseases and the efficacy of drugs. Currently, the FDA has approved more than 150 LDT detection methods based on plasma proteins. These demonstrate the clinical application value of plasma proteomics. In general, plasma proteomics research is not only of great value in the discovery of clinical disease markers and disease risk prediction, but also in drug development, such as new drug target discovery, drug targeting performance confirmation, drug efficacy and adverse reaction monitoring, pharmacodynamic marker discovery, new drug indication expansion, responder characteristic analysis, etc. It plays an irreplaceable role.

[0003] In 2001, the International Human Proteome Organization was established. The first human proteome project launched by HUPO in 2002 was the "Human Plasma Proteome Project". In the 10 years from the launch of the Human Plasma Proteome Project in 2002 to its completion in 2011, dozens of scientific research units around the world participated and hundreds of related papers were published. After integrating the massive research results from around the world, through methods such as high-abundance protein removal strategies and multi-dimensional separation strategies to increase the mass spectrometry loading time by several times, the protein identification results of multiple technical routes, multiple laboratories, and multiple batches of experiments were merged, and only 1,929 plasma proteins were finally confirmed. This is because the analysis of the human plasma proteome is extremely challenging, and high-abundance proteins seriously affect the detection of low-abundance proteins. In plasma, albumin alone accounts for half of the total plasma protein mass, immune proteins account for about 25%, and the top 22 proteins in abundance account for more than 99% of the total plasma protein mass. Plasma samples were analyzed by proteomics. In a single mass spectrometry analysis, the number of plasma proteins identified by mass spectrometry for a plasma sample was about 300. After being processed with a commercial plasma high-abundance protein removal kit, the number of plasma proteins identified by mass spectrometry was generally between 400 and 700 proteins, and more than 80% of these identification results were medium- and high-abundance functional proteins in the blood. However, low-concentration proteins are often related to the evolution of the disease and are very valuable biomarkers.

[0004] In other words, the main problem faced by blood proteomics research using mass spectrometry as the main detection method is that due to the serious interference of high-abundance proteins in the blood during mass spectrometry detection, the mass spectrometer repeatedly collects repeated signals, resulting in the inability to detect low-abundance proteins. Conventional commercial high-abundance removal methods can only partially remove more than a dozen high-abundance proteins, and the residual high-abundance proteins will still interfere with the detection of extremely low-content proteins. In addition, since high-abundance proteins often have transport functions, the removal of high-abundance proteins will also be accompanied by the removal of many valuable low-abundance plasma proteins. Therefore, in the sample processing method before mass spectrometry detection, if the low-abundance plasma proteins can be obtained more effectively, it is crucial for plasma proteome analysis. Summary of the invention

[0005] In one aspect, the present invention provides a method for selectively enriching low-abundance proteins from a sample containing multiple proteins, comprising the steps of:

[0006] 1) adding carbon nanotubes modified with benzophenonesulfonic acid to the sample, so that a portion of the multiple proteins are adsorbed onto the surface of the carbon nanotubes; and

[0007] 2) Separating the carbon nanotubes from the sample.

[0008] In some embodiments, the proteins with smaller molecular weight among the multiple proteins in step 1) can enter the interior of the carbon nanotube and adsorb on the inner surface of the carbon nanotube, while the proteins with larger molecular weight among the multiple proteins cannot enter the interior of the carbon nanotube and adsorb on the outer surface of the carbon nanotube.

[0009] In some embodiments, the method further comprises the step: 3) separating the protein adsorbed by the carbon nanotubes from the carbon nanotubes to obtain an enriched sample enriched with the low-abundance protein.

[0010] In some embodiments, the carbon nanotubes are multi-walled carbon nanotubes.

[0011] In some embodiments, the carbon nanotubes are helical multi-walled carbon nanotubes.

[0012] In some embodiments, the carbon nanotubes are magnetic carbon nanotubes.

[0013] In some embodiments, the magnetic carbon nanotubes are 3 O 4 Imparts magnetism.

[0014] In some embodiments, the inner diameter of the carbon nanotubes is 20-50 nm.

[0015] In some embodiments, the sample is a biological sample, such as plasma or serum.

[0016] In some embodiments, in step 3), a detergent is used to separate the protein adsorbed by the carbon nanotubes from the carbon nanotubes; preferably, the detergent is 1-dodecyl-3-methylimidazole chloride (C12Im-Cl).

[0017] In another aspect, the present invention provides a method for mass spectrometry identification of plasma proteins in a plasma sample, comprising:

[0018] 1) preparing an enriched plasma sample enriched with low-abundance proteins from the plasma sample by the above method;

[0019] 2) treating the enriched plasma sample with a protease; and

[0020] 3) Determining the type and / or content of plasma proteins in the enriched plasma sample by mass spectrometry analysis.

[0021] In some embodiments, the protease is a mixture of trypsin and the intracellular protease Lys-C.

[0022] In some embodiments, the mass spectrometry analysis is performed using tandem mass spectrometry including a primary mass spectrometry and a secondary mass spectrometry, preferably using DIA mass spectrometry.

[0023] In some embodiments, the primary mass spectrometry scanning range is set to 400-1100 m / z; and the secondary mass spectrometry scanning window number is set to 40.

[0024] In another aspect, provided herein is a method for diagnosing a disease in a subject, comprising identifying the type and / or amount of a plasma protein in plasma from the subject using the above method.

[0025] In some embodiments, the disease is lung cancer, such as early-stage lung cancer or advanced-stage lung cancer.

[0026] The method disclosed in this article for preparing plasma proteome samples using magnetic helical multi-walled carbon nanotubes modified with pyrenesulfonic acid uses a new non-ionic surfactant C12Im-Cl to remove non-specifically adsorbed proteins, and combines it with a high-resolution mass spectrometry data acquisition method to achieve the qualitative and quantitative analysis of more than 2,000 plasma proteins in a single mass spectrometry injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a scanning electron micrograph of magnetic helical multi-walled carbon nanotubes modified with benzophenonesulfonic acid.

[0028] Figure 2 This is a photo of helical multi-walled carbon nanotubes and magnetic helical multi-walled carbon nanotubes modified with pyrenebutyric acid after the action of a magnet.

[0029] Figure 3The mass-to-charge ratio distribution of the primary precursor ions of the plasma proteome samples is shown.

[0030] Figure 4 The figure shows the comparison of different plasma sample preparation methods and mass spectrometry acquisition modes on the qualitative quantity of plasma proteins (n=3).

[0031] Figure 5 The mass spectrometry identification quantity distribution of plasma proteins after treating different plasma volume samples with h-MWCNTs@Fe3O4 and h-MWCNTs@Fe3O4 modified with pyrenebutyric acid is shown.

[0032] Figure 6 Pearson correlation diagram of three technical replicates of the method of the present invention.

[0033] Figure 7 This is a distribution diagram of the coefficient of variation of protein quantification obtained by three technical replicates of the method of the present invention.

[0034] Figure 8 The unsupervised cluster analysis results of the present invention method applied to 5 biological replicates of plasma from healthy volunteers, early lung cancer patients, and advanced lung cancer patients are shown. A, B, and C represent the three groups of healthy volunteers, early lung cancer patients, and advanced lung cancer patients, respectively. Each point in the figure is a dimensionality reduction representation of all plasma protein quantitative values ​​identified by biological replicates within a group. DETAILED DESCRIPTION

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0036] Unless the context clearly indicates otherwise, the term "or" refers to a single element of the listed alternative elements and the term "and / or" refers to any one, any two, any three, any more or all of the listed alternative elements.

[0037] The term "comprising" or "including" means including the elements, integers or steps mentioned, but does not exclude any other elements, integers or steps. In the text, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the situation consisting of the elements, integers or steps mentioned. For example, when it is mentioned that a mixture of several components is "comprising", it is also intended to cover the mixture consisting of these components.

[0038] "Low-abundance protein" refers to one or more proteins with relatively less content (or concentration) in a sample including multiple proteins in this article. This term is relative to the term "high-abundance protein", indicating the relative content of different protein species in the same sample. In some examples, the content of a low-abundance protein or the total content of two or more low-abundance proteins can be less than 10% (wt) of the total protein amount in the sample, for example, less than 5%, 1%, 0.1%, 0.01% or less of the total protein amount. In other examples, the multiple proteins in the sample can be sorted by content (such as wt), and the protein below the median content can be considered as "low-abundance protein", or the protein below the first quartile or even the third quartile content is considered as "low-abundance protein". The remaining protein after removing the low-abundance protein in the sample including multiple proteins can be considered as "high-abundance protein", and vice versa, that is, the remaining protein after removing the high-abundance protein in the sample including multiple proteins can be considered as "low-abundance protein".

[0039] "Selective enrichment of low-abundance proteins" refers in this article to a sample including multiple proteins, after being treated, the proportion of one or more low-abundance proteins of interest in the total protein content of the treated sample is increased. In some examples, selective enrichment of low-abundance proteins can be achieved by removing relatively more high-abundance proteins in the sample. In other examples, selective enrichment of low-abundance proteins can be achieved by recovering relatively more low-abundance proteins in the sample. For example, after the sample is treated, a certain protein or several low-abundance proteins in the sample increase from 1% (wt) of the total protein in the sample to 2%, 5%, 10% or more. "Selective enrichment of low-abundance proteins" does not exclude the possibility of partial loss of low-abundance proteins when processing the sample, but the proportion of total protein content is improved.

[0040] "Carbon nanotubes (CNTs)" can be regarded as nanoscale cylindrical molecules composed of a single layer or multiple layers of carbon atoms (graphene) rolled up. Those that only include a single layer of carbon atoms are called single-walled carbon nanotubes (SWCNTs), and those that include multiple concentrically interconnected single-walled carbon nanotubes are called multi-walled carbon nanotubes (MWCNTs). Their diameters range from 1 to 100 nm. The carbon atoms in carbon nanotubes are mainly sp2 hybridized and bonded to the surrounding carbon atoms to form a large π delocalized system. Due to its unique structure, it has many unique properties, such as electrical conductivity, thermal conductivity, mechanical properties and chemical stability. "Helical carbon nanotubes" refer to carbon nanotubes with a helical shape, which may be caused by the presence of non-carbon six-ring structures (such as carbon five rings and carbon seven rings) in carbon nanotubes. In addition, the carbon nanotubes used in this article can also be modified by sodium 1-pyrenesulfonate modification, as well as by loading ferrosoferric oxide (Fe 3 O 4 ) imparts magnetism.

[0041] The protein enrichment method provided herein realizes the enrichment of low-abundance proteins in plasma and the selective removal of high-abundance proteins through the ultra-large specific surface area and spatial confinement of magnetic spiral multi-walled carbon nanotubes modified by pyrenesulfonic acid, effectively improving the relative abundance of low-abundance proteins in plasma samples, avoiding the signal suppression of low-abundance proteins by high-abundance proteins in plasma during mass spectrometry analysis, and significantly improving the depth of plasma protein identification under the condition of mass spectrometry injection of a single sample. Moreover, the sample processing time in the present invention is as short as 4 hours, which is more efficient than other conventional technologies that require a sample processing time of about 12-24 hours, and significantly improves the processing throughput of plasma proteome samples.

[0042] Magnetic helical multi-walled carbon nanotubes have been successfully applied to energy storage, biosensing and drug delivery due to their long-range ordered sp2 hybridization structure, large specific surface area and spatial confinement of tube diameter. Among them, sp2 hybridization refers to the process of hybridization of one ns orbital and two np orbitals in the same electron layer of an atom. After the atom undergoes sp2 hybridization, the above-mentioned ns orbital and np orbital will be transformed into three equivalent atomic orbitals, called "sp2 hybrid orbitals". The symmetry axes of the three sp2 hybrid orbitals are on the same plane, and the angles between them are all 120°. The sp2 structure can maintain the mechanical properties of carbon nanotubes and inhibit the self-aggregation of carbon nanotubes during use. However, there are certain limitations when using single-component helical carbon nanotube materials, such as weak electrochemical activity and easy agglomeration, which greatly limits their application. Therefore, functional modification is crucial to expand its application. The present invention utilizes the π-π interaction between carbon nanotubes and 1-pyrene sulfonate having the same π-π conjugated structure to achieve functional modification of carbon nanotubes. This method can improve the dispersibility of carbon nanotubes and increase their selective adsorption performance for other biological molecules while maintaining the bulk structure and excellent performance of carbon nanotubes.

[0043] After entering the biological environment, the magnetic helical multi-walled carbon nanotube material modified by pyrenesulfonic acid will form a protein corona with proteins quickly and non-concentration-dependently. This property can be used for the specific enrichment of low-abundance proteins in the blood. It is currently known that among the top 100 proteins with the highest abundance of high-abundance proteins in plasma, more than 50% of the proteins have a molecular weight greater than 50,000. Therefore, by utilizing the molecular interception effect of the ultra-large specific surface area and pore size of the helical magnetic multi-walled carbon nanotube modified by pyrenesulfonic acid, it will be possible to achieve specific enrichment of low-abundance proteins in plasma while selectively removing most of the high-abundance proteins. The magnetic properties of the material can then be used to achieve rapid separation of samples, which can be applied to the sample processing of plasma proteomes.

[0044] 1-Dodecyl-3-methylimidazolium chloride (C12Im-Cl) is a type of ionic liquid widely used in the extraction of hydrophobic membrane proteins. This paper first used C12Im-Cl to remove nonspecific high-abundance proteins adsorbed on the surface of helical multi-walled carbon nanotubes modified with pyrenesulfonic acid, avoiding the loss of low-abundance samples caused by desalting after the use of conventional surfactants. Through high-resolution mass spectrometry acquisition, the depth of plasma proteome identification was improved.

[0045] The present invention is further described in detail below by means of specific examples. The following examples are illustrative and are not intended to be limiting.

[0046] Instruments and reagents

[0047] Perylenetetracarboxylic acid dianhydride, 1-pyrenesulfonic acid sodium salt, ammonia water, ethylene glycol, trisodium citrate dihydrate, and anhydrous sodium acetate were purchased from Sigma-Aldrich. Ultramicro spectrophotometer Nano Drop, mass spectrometer Orbitrap Exploris 480, constant temperature mixer and other equipment were purchased from Thermo Fisher Scientific, and refrigerated centrifuge was purchased from Beijing Ji Aim Technology Co., Ltd. Example 1 Preparation of helical multi-walled carbon nanotubes

[0048] Dissolve 400 mg of 3,4,9,10-perylenetetracarboxylic dianhydride powder in 5 mL of deionized water, stir at room temperature for 1 hour, add 1 mL of ammonia water, quickly transfer the red solution to a 10 mL reactor, place the reactor in a preheated oven, and perform a hydrothermal reaction at 100 ° C for 24 hours. After the reaction is completed, centrifuge at 1200 g for 15 minutes, remove the supernatant, and wash the solid matter obtained by centrifugation with deionized water for three times. Finally, place the precipitate in a freeze dryer to dry. Take 50 mg of the dried product and transfer it to a tube furnace and condense it with an inert N 2 The air in the quartz tube was replaced by purging for 30 minutes, a nitrogen environment was maintained, and the temperature was raised to 600°C at a rate of 5°C / min and kept at a constant temperature for 6 hours. After turning off the tubular furnace, the temperature in the furnace was allowed to naturally drop to room temperature. The solid powder obtained was helical multi-walled carbon nanotubes (h-MWCNTs).

[0049] Example 2 Synthesis of Magnetized Helical Multi-walled Carbon Nanotubes Modified with Benzoylsulfonic Acid

[0050] Add 30 mg h-MWCNTs, 160 mg FeCl 3 6H 2O and 30 mg of trisodium citrate dihydrate, ultrasonically dissolve for 30 min, add 700 mg of anhydrous sodium acetate, and mechanically stir for 30 min at room temperature. Slowly drop 0.3 mL of concentrated ammonia (28%, v / v) and stir to mix. Transfer to a stainless steel reactor and place in an electric heating furnace preheated to 220 ° C for 9 hours. After the reaction is completed, cool to room temperature and separate with a magnet to obtain h-MWCNTs@Fe 3 O 4 The product was washed and dried under vacuum at 60 °C. 3 O 4 Add 40 mL of deionized water and react with ultrasound for 30 minutes. Add 20 mg of 1-pyrenesulfonic acid sodium salt (PyS), 20 mg of sodium hydroxide, and 125 μL of hydrazine hydrate to the suspension, stir vigorously at 80 ° C for 24 hours, remove the supernatant by magnetic separation, and wash 3 times with 50 mL of deionized water. The black product is the magnetized spiral multi-walled carbon nanotubes modified with pyrenesulfonic acid.

[0051] h-MWCNTs@Fe modified with benzophenonesulfonic acid 3 O 4 Characterization: Scanning electron microscopy showed that the structure of the helical nanotubes modified with benzosulfonic acid was the same as that reported previously ( Figure 1 ). The helical nanotubes modified with benzophenonesulfonic acid are used to modify Fe 3 O 4 After that, it showed good superparamagnetism. The unmodified magnetic helical nanotubes did not show magnetic separation under the action of a magnet, while the magnetic helical nanotubes modified with pyrenesulfonic acid showed superparamagnetism under the action of a magnet ( Figure 2 ).

[0052] Example 3 h-MWCNTs@Fe modified with benzophenonesulfonic acid 3 O 4 For enrichment of plasma proteins

[0053] 500 μg of h-MWCNTs@Fe modified with benzophenonesulfonic acid was added 3 O 4After being dispersed in 100 μL incubation buffer (10 mM Tris (pH = 7.4), 150 mM KCl, 0.05% (1-dodecyl-3-methylimidazolium chloride, C12Im-Cl), ultrasonic for 10 seconds, 100 μL plasma was added to the system and incubated at 37 ° C for 1 hour. After the incubation was completed, an external magnetic field was applied to remove the supernatant solution, and the obtained precipitate was washed 3 times with 200 μL incubation buffer, and the incubation buffer was discarded. 40 μL of 1% C12Im-Cl, 10 mM TECP, 40 mM CAA, 50 mM TEAB enzyme digestion buffer (pH 8.5) was added, reacted at 95 ° C for 5 minutes, 160 μL of anhydrous methanol solution was added, and the reaction was shaken for 15 minutes. After magnetic separation to remove the supernatant, h-MWCNTs@Fe was washed with 80% methanol (v / v). 3 O 4 After three times, 50 μL of 50 mM ammonium bicarbonate solution was used to redisperse h-MWCNTs@Fe 3 O 4 , then add 250 ng of sequencing-grade trypsin and Lys-C, incubate at 37°C for 1.5 h. Centrifuge at 10,000 g for 2 minutes, collect the supernatant, and perform subsequent mass spectrometry analysis.

[0054] Example 4 Liquid chromatography-high resolution mass spectrometry analysis

[0055] The heat-dried plasma proteome sample prepared in Example 3 was re-dissolved in 0.1% formic acid aqueous solution, and 500 ng was taken for mass spectrometry detection. Data acquisition was performed using a ThermoScientific nanoliter liquid chromatography tandem high-resolution mass spectrometer (OrbitrapExploris 480). The chromatographic method column was: C 18 Packing, 20cm×150μm inner diameter. Mobile phase A is 0.1% formic acid, mobile phase B is acetonitrile and 0.1% formic acid, flow rate is 600nL / min. Liquid phase gradient is: 0 to 5min (5-10% B); 10 to 58min (12-30% B); 58 to 68min (30-45% B); 68 to 70min (45-95% B); 70 to 78min (95% B). Data acquisition mode: DIA, ion source: NSI, ion source voltage: 2.1kV. The primary mass spectrometer scanning range is set to 400-1100m / z, scanning resolution: 120,000; secondary scanning window number: 40, secondary scanning resolution: 30,000. Ion fragmentation mode: high energy collision dissociation mode (HCD), fragmentation energy: 25, 27, 30; secondary scan automatic gain control (AGC): 1E6, maximum injection time: 40ms.

[0056] Data analysis: DIA-NN (v1.8) was used for retrieval. The search parameters were as follows: the database was Swissprot_human protein database (20588 sequences); the enzyme digestion method was set to Trypsin / P, and the maximum number of missed cleavage sites was set to 2; the mass error range of the primary and secondary mass spectrometry was 20ppm. The fixed modification was set to Carbamidomethyl (C); the variable modification was Oxidation (M), Acetyl (N-terminus).

[0057] Results: In this paper, 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) was used as the h-MWCNTs@Fe modified with pyrenesulfonic acid. 3 O 4 Detergents that adsorb plasma proteins non-specifically. This reagent not only has a strong ability to remove non-specific adsorbed proteins, but is also compatible with mass spectrometry. It can directly enter the sample into mass spectrometry analysis without desalting, avoiding the loss of low-abundance peptides caused by desalting during sample preparation, and effectively reducing the interference of conventional detergents such as sodium dodecyl sulfate (SDS) and sodium deoxycholate (SDC) on mass spectrometry detection. According to the m / z distribution characteristics of plasma sample peptide ions, such as Figure 3 As shown, the primary scanning range of the DIA mass spectrometer is changed from the conventional 350-1400 to 400-1100, which improves the effectiveness of the secondary spectrum acquisition quality and increases the qualitative and quantitative ratio of the spectrum. In addition, the dynamically changing parent ion isolation window setting is adopted to further improve the accuracy of secondary spectrum identification. Therefore, while using a new detergent to remove non-specific protein adsorption and reduce the loss of low-abundance peptides, combined with a new narrow mass spectrometry scanning range, the utilization rate of plasma protein spectra is effectively improved. Compared with untreated plasma, commercial plasma high abundance removal methods (such as Top14), h-MWCNTs@Fe3O4 high abundance removal, and SDS / SDC detergents, the h-MWCNTs@Fe3O4 modified with pyrenesulfonic acid used in the present invention is 3 O 4 The combination of C12Im-Cl detergent and improved mass spectrometry acquisition method significantly improved the protein identification depth of a single plasma sample. In a single injection of a plasma sample, more than 2000 plasma proteins were qualitatively and quantitatively analyzed, such as Figure 4 shown.

[0058] Example 5 h-MWCNTs@Fe modified with benzophenonesulfonic acid 3 O 4 Methodological evaluation of enriched plasma proteins

[0059] 5.1 h-MWCNTs@Fe modified with benzophenonesulfonic acid 3 O 4 In the presence of h-MWCNTs@Fe 3 O 4 (without pyrenesulfonic acid modification) Under the same mass spectrometry detection conditions and SDS surfactant to remove non-specific protein adsorption, the number of protein identifications of the method of the present invention is better than that of h-MWCNTs@Fe 3 O 4 method( Figure 5 ).

[0060] 5.2 The Pearson correlation coefficients of the three technical replicates of the method of the present invention were all greater than 0.9 ( Figure 6 ), 70% of the identified plasma proteins (black circles in the figure) had a coefficient of variation of less than 20% ( Figure 7 ), indicating that the results of plasma protein enrichment by this method are stable and have good reproducibility.

[0061] 5.3 h-MWCNTs@Fe modified with benzophenonesulfonic acid 3 O 4 The plasma proteome samples prepared from the materials were combined with the mass spectrometry detection method of the present invention to detect the qualitative and quantitative information of plasma proteins. Unsupervised cluster analysis (using R language software package) was used to significantly distinguish the plasma proteome samples of 5 biological replicates of healthy volunteers, early lung cancer patients, and late lung cancer patients ( Figure 8 ), indicating that this method has a good ability to distinguish individual differences.

[0062] The present invention adopts h-MWCNTs@Fe modified with benzoylsulfonic acid 3 O 4 Enriched plasma proteins. Compared with existing h-MWCNTs, this type of material has a larger specific surface area, adsorption efficiency and spatial confinement effect. It can not only adsorb low-abundance plasma proteins by modifying with pyrenesulfonic acid and forming a protein corona on the surface of the nanomaterial, but also selectively retain large molecular weight and high-abundance proteins through the pore size of the nanotube (spatial confinement effect). Combined with the new C12Im-Cl detergent cleaning, it effectively avoids the interference of high-abundance proteins in plasma on the identification of low-abundance proteins during mass spectrometry analysis, and reduces the loss of low-abundance peptides before mass spectrometry detection. In order to improve the efficiency of DIA mass spectrometry data acquisition, the primary mass spectrometry scanning range and the parent ion isolation window settings are optimized according to the molecular weight distribution characteristics of plasma sample peptides, which significantly improves the number of identifications of single plasma samples and the credibility of quantitative data.

[0063] The method of the present invention has high detection sensitivity and is simpler to operate. The processing time of plasma proteome samples only takes 4 hours (the existing technology takes 12 to 24 hours), which can greatly improve the processing throughput of plasma samples. The number of qualitative and quantitative proteins in a single plasma sample detected by mass spectrometry is nearly 2,000, which is of great significance for the screening research of plasma protein markers for major diseases such as breast cancer.

[0064] In view of the many possible embodiments to which the principles of the present disclosure may be applied, those skilled in the art should recognize that the illustrated embodiments are exemplary only and should not be taken as limiting the scope of the invention.

Claims

1. A method for selectively enriching low-abundance proteins from a sample containing multiple proteins, comprising the steps of: 1) adding carbon nanotubes modified with benzophenonesulfonic acid to the sample, so that a portion of the multiple proteins are adsorbed onto the surface of the carbon nanotubes; and 2) separating the carbon nanotubes from the sample, in, The carbon nanotubes modified with pyrenesulfonic acid are magnetic helical multi-walled carbon nanotubes modified with pyrenesulfonic acid. Wherein, the inner diameter of the carbon nanotube is 20-50nm, The pyrenesulfonic acid-modified carbon nanotubes are obtained by functionalizing the carbon nanotubes through the π-π interaction between the carbon nanotubes and 1-pyrenesulfonic acid salts having a π-π conjugated structure.

2. The method as claimed in claim 1, wherein in step 1), proteins with smaller molecular weight among the multiple proteins can enter the interior of the carbon nanotube and adsorb on the inner surface of the carbon nanotube, and proteins with larger molecular weight among the multiple proteins cannot enter the interior of the carbon nanotube and adsorb on the outer surface of the carbon nanotube.

3. The method according to claim 1 or 2, further comprising the step of: 3) separating the protein adsorbed by the carbon nanotubes from the carbon nanotubes to obtain an enriched sample enriched with the low-abundance protein. The method according to claim 1 or 2, wherein the magnetic carbon nanotubes are endowed with magnetic properties by Fe3O4.

5. The method of claim 1 or 2, wherein the sample is a biological sample. The method of claim 1 or 2 , wherein the sample is plasma or serum.

7. The method according to claim 1 or 2, wherein in step 3), a detergent is used to separate the protein adsorbed by the carbon nanotubes from the carbon nanotubes.

8. The method of claim 7, wherein the detergent is 1-dodecyl-3-methylimidazole chloride.

9. A method for mass spectrometry identification of plasma proteins in a plasma sample, comprising: 1) preparing an enriched plasma sample enriched with low-abundance proteins from the plasma sample by the method according to any one of claims 1 to 8; 2) treating the enriched plasma sample with a protease; as well as 3) Determining the type and / or content of plasma proteins in the enriched plasma sample by mass spectrometry analysis.

10. The method of claim 9, wherein the protease is a mixture of trypsin and the intracellular protease Lys-C.

11. The method according to claim 9 or 10, wherein the mass spectrometry analysis is performed using a tandem mass spectrometer comprising a primary mass spectrometer and a secondary mass spectrometer.

12. The method according to claim 9 or 10, wherein the mass spectrometry analysis is performed using DIA mass spectrometry.

13. The method of claim 11, wherein the primary mass spectrometry scanning range is set to 400-1100 m / z; and the secondary mass spectrometry scanning window number is set to 40.

14. Use of pyrenesulfonic acid-modified carbon nanotubes in the preparation of a preparation for diagnosing lung cancer in a subject, wherein the pyrenesulfonic acid-modified carbon nanotubes are pyrenesulfonic acid-modified carbon nanotubes as defined in any one of claims 1 to 4.

15. The use according to claim 14, wherein the lung cancer is early stage lung cancer or late stage lung cancer.

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