CN115951070A - Method for detecting protein in sample - Google Patents
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- CN115951070A CN115951070A CN202310083005.4A CN202310083005A CN115951070A CN 115951070 A CN115951070 A CN 115951070A CN 202310083005 A CN202310083005 A CN 202310083005A CN 115951070 A CN115951070 A CN 115951070A
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Abstract
The invention provides a method for detecting protein in a sample, which is characterized by comprising the following steps: s1, mixing the sample with magnetic nanoparticles to obtain protein-bound magnetic nanoparticles; s2, sequentially carrying out reduction treatment, alkylation treatment, washing treatment and enzyme digestion treatment on the protein-bound magnetic nanoparticles to obtain an enzyme digestion product; s3, performing mass spectrum detection on the enzyme digestion product; the magnetic nanoparticles comprise ferroferric oxide nanoparticles and a covalent organic framework formed on the ferroferric oxide nanoparticles, wherein the covalent organic framework is obtained by reacting 1,3, 5-trimethylresorcinol and 1, 4-diaminobenzene. Through the technical scheme, the qualitative depth, the quantitative stability, the reliability and the time efficiency of the nano material in the clinical proteomics application are improved.
Description
Technical Field
The present application relates to the field of biotechnology, in particular, to a method for detecting a protein in a sample.
Background
Plasma is an easily obtained minimally invasive sample that records the real-time status of the body and feeds back the integrated results of different lifestyle, disease, treatment or other related changes directly to the subject. The plasma contains a large amount of tissue leakage proteins, signal molecules and cytokines besides functional proteins such as human serum albumin, apolipoprotein, coagulation cascade protein and the like. Approximately 10546 proteins were recorded in the plasma proteome database (www. Plasma proteome studies are crucial for biomarker development for disease diagnosis, risk prediction, prognostic monitoring, and treatment effect assessment.
Over the last few years, there has been a great deal of research devoted to finding disease-related biomarkers from plasma samples. Proteomics of plasma and liver biopsies have been analyzed to find biomarkers that predict future events of relevance and mortality from alcohol-related liver disease. High-throughput, unbiased quantitative proteomics has also been employed to investigate the relationship between plasma protein levels and subclinical atherosclerosis. The discovery of plasma protein biomarkers is particularly important for patients with neurological disorders where cerebrospinal fluid and brain tissue samples are difficult to obtain. There have also been attempts to develop biomarkers by analyzing the plasma proteome of alzheimer's disease. But advances have been limited due to the lack of depth of proteomics.
The high abundance of protein inhibition in plasma samples makes it very difficult to detect plasma biomarkers at ng/ml level. To improve the detectability of low-abundance proteins, various pretreatment strategies have been established, including immune or affinity-based high-abundance removal methods, liquid phase separation methods, and sub-proteome enrichment methods. In a prior study, 1544 plasma proteins and 258 new proteins were identified by high pH RPLC separation and LC-MS/MS analysis.
Despite the increased depth of proteomics, this strategy does not meet the demand for high throughput analysis of clinical samples.
The nano material is used for carrying medicine into a target area of a body for diagnosis or treatment, and the nano material is found to adsorb multiple layers of protein on the surface of the nano material to form a protein corona when entering a biological environment. The nanomaterial interacts with blood plasma to form a protein corona. The high-abundance proteins are initially bound to the nanomaterial surface and subsequently replaced by high-affinity low-abundance proteins (Vroman effect). The protein crown can reduce the dynamic range of protein concentration, enrich low-abundance and medium-abundance proteins and increase the detection depth of plasma proteins.
In recent years, a great deal of research from the field of materials has shown that nanomaterials with different modifying groups and different substrates have different protein corona characteristics. By characterizing the protein corona, one group of proteins showing significant differences in breast and prostate cancer patients and controls was identified. While the use of nanomaterials can be used to conduct disease proteomics studies, their qualitative depth, quantitative stability, reliability and time efficiency in clinical proteomics applications still need to be improved.
Disclosure of Invention
The invention aims to improve the qualitative depth, quantitative stability, reliability and time efficiency of nano materials in clinical proteomics application.
In order to achieve the above object, the present invention provides a method for detecting a protein in a sample, comprising the steps of: s1, mixing the sample with magnetic nanoparticles to obtain protein-bound magnetic nanoparticles; s2, sequentially carrying out reduction treatment, alkylation treatment, washing treatment and enzyme digestion treatment on the protein-bound magnetic nanoparticles to obtain an enzyme digestion product; s3, performing mass spectrum detection on the enzyme digestion product; the magnetic nanoparticles comprise ferroferric oxide nanoparticles and covalent organic frameworks formed on the ferroferric oxide nanoparticles, wherein the covalent organic frameworks are covalent organic frameworks obtained by reacting 1,3, 5-trimethylresorcinol and 1, 4-diaminobenzene.
Through the technical scheme, the qualitative depth, the quantitative stability, the reliability and the time efficiency of the nano material in the clinical proteomics application are improved.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
fig. 1 is a TEM image of DMB beads.
FIG. 2 is a graph showing the results of protein identification of the same plasma after high-abundance protein removal (removal of 2 and 14 high-abundance proteins), hexapeptide ligand enrichment and DMB bead enrichment.
FIG. 3 is a graph showing the results of testing DMB beads for their ability to enrich for proteins (protein-identifying species) at concentrations ranging from. Mu.g/L and ng/L.
Fig. 4 is a graph showing the results of testing DMB bead-enriched proteins (versus theoretical abundance).
FIG. 5 shows the amount of protein identified by the enzyme digestion on the DMB bead magnetic bead compared to the solution digestion at different enzyme digestion times.
FIG. 6 is a graph showing the comparison of the results of plasma protein quantification, in which the horizontal axis represents the gradient of E.coli protein and the vertical axis represents the fold change in the quantification of human plasma protein.
FIG. 7 is a graph showing a comparison of the results of E.coli protein quantification, in which the horizontal axis represents the amount of E.coli protein gradient and the vertical axis represents the fold change in E.coli protein quantification.
FIG. 8 is the effect of DMB beads and literature reported consumables on the number of protein detected.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are given by way of illustration and explanation only, not limitation.
The invention provides a method for detecting protein in a sample, which is characterized by comprising the following steps: s1, mixing the sample with magnetic nanoparticles to obtain protein-bound magnetic nanoparticles; s2, sequentially carrying out reduction treatment, alkylation treatment, washing treatment and enzyme digestion treatment on the protein-bound magnetic nanoparticles to obtain an enzyme digestion product; s3, performing mass spectrum detection on the enzyme digestion product; the magnetic nanoparticles comprise ferroferric oxide nanoparticles and a covalent organic framework formed on the ferroferric oxide nanoparticles, wherein the covalent organic framework is obtained by reacting 1,3, 5-trimethylresorcinol and 1, 4-diaminobenzene.
Optionally, wherein the magnetic nanoparticles have a particle size of 0.1-0.3 μm.
Optionally, wherein, in the magnetic nanoparticles, the content of the covalent organic skeleton is 2 to 4 parts by weight per part by weight of the ferroferric oxide nanoparticles.
Optionally, wherein the method further comprises preparing the magnetic nanoparticles by: SS1, dissolving ferric trichloride hexahydrate and sodium acetate in ethylene glycol to obtain a homogeneous transparent solution; the weight ratio of ferric trichloride hexahydrate to sodium acetate is 1; the dosage of the ethylene glycol is 15-25mL relative to 1g of ferric trichloride; SS2, carrying out a first solvothermal reaction on the homogeneous transparent solution at the temperature of 150-250 ℃ for 4-24 hours to obtain black powder; SS3, mixing the black powder, 1,3, 5-trimethylresorcinol and 1, 4-diaminobenzene in a weight ratio of 1:1-2:1-2 in ethanol and carrying out a second solvothermal reaction at 150-250 ℃ for 20-80 hours; the amount of ethanol used is 15-25mL relative to 1g of the black powder.
Optionally, wherein the sample is whole blood, plasma, serum, or cerebrospinal fluid.
Optionally, wherein the sample is mixed with the magnetic nanoparticles after dilution by a factor of 1-3.
Optionally, wherein the amount of the magnetic nanoparticles is 0.05-0.1mg relative to 1mL of the diluted sample.
Optionally, wherein the reducing treatment conditions comprise: the reducing agent is tris (2-carboxyethyl) phosphine (TCEP), the concentration of the reducing agent is 0.01-0.02 mu M, the time is 10-30 minutes, and the temperature is 92-98 ℃; the alkylation conditions include: the alkylating agent is Iodoacetamide (IAA), the concentration of the alkylating agent is 0.025-0.050 μ M, the time is 10-30 minutes, and the temperature is 10-30 ℃.
Optionally, wherein the washing treatment comprises washing with an acetonitrile solution containing 70 to 90 vol% acetonitrile in water; the enzymatic treatment comprises enzymatic cleavage using trypsin.
Optionally, the mass spectrometric detection of the cleavage product comprises qualitative detection and/or quantitative detection.
The present invention will be described in further detail below with reference to examples. The raw materials used in the examples are all available from commercial sources.
Example 1
36 plasma samples were tested for inclusion (multiple system atrophy, MSA, n =18; non-neurological disease control group, HC, 18). These samples were collected from 11 months to 12 months in 2020 to 2021. MSA patients were diagnosed by two medical professionals based on established consensus criteria. Selected age and gender matched control group subjects were from a healthy screening population. Those samples that were not age and gender comparable were excluded. Whole blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes. Centrifuge at 4000g for 10min at 4 ℃. The plasma obtained was stored in equal amounts at-80 ℃. Ferric chloride hexahydrate was obtained from Sigma-Aldrich (st. Louis, MO, USA). 1,3, 5-Triacetoresorcinol, ethylene glycol and 1, 4-diaminobenzene (Pa-1) were supplied by Sigma-Aldrich (St. Louis, mo., USA) and Aladdin industries, inc. (Shanghai, china), respectively. Tris (2-carboxyethyl) phosphine (TCEP), iodoacetamide (IAA) and tetraethylammonium bromide (TEAB) were purchased from Sigma-Aldrich (st. Louis, MO, USA).
The preparation of magnetic nanoparticles was carried out as follows. Briefly, 1.0 gram of FeCl 3 ·6H 2 O,3.6g of 1, 6-hexadiamine and 4.0g of CH 3 COONa was dissolved in 30ml of an ethylene glycol solution, stirred with ultrasound with strong magnetism for 40 minutes to obtain a uniform transparent solution, and then sealed in an autoclave to conduct a solvothermal reaction at 200 ℃ for 6 hours. Washing with clear water and ethanol for 3 times to obtain black powder, and drying at 50 deg.C for 6 hr to obtain magnetic Fe 3 O 4 。
60mg of magnetic Fe 3 O 4 Dispersed in 30mL of anhydrous ethanol containing 1,3, 5-trimethylacylresorcinol and 1, 4-diaminobenzene. The mixture was sonicated for 35 minutes in an autoclaveHeat to 180 ℃ for 48 hours. The obtained magnetic nanoparticles were washed with ethanol, water and ethanol, respectively, and dried at 50 ℃ for 6h. Morphology and structure observations of magnetic nanoparticles (i.e., DMB beads) were performed on hitachi 4800 field emission scanning electron microscope (FE-SEM) and FEI Tecnai G20 Transmission Electron Microscope (TEM). A TEM image of the DMB bead is shown in fig. 1.
An amount of plasma was diluted with PBS buffer (pH 7.4) and incubated with magnetic nanoparticles at 37 ° for 30min. Unbound protein was discarded and the magnetic nanoparticles were washed three times with PBS buffer (pH 7.4). The buffer was replaced with lysis buffer (50Mm TEAB,10mM TCEP,50Mm IAA). The magnetic material was reduced and alkylated at 95 ℃ for 10min and then incubated with 80% acetonitrile for 20min. After replacing the solvent with 50mM ammonium bicarbonate solution, trypsin was added at 37 ℃. After digestion for 4 hours, the reaction was quenched with 0.1% TFA buffer. The cleaved peptide was used for LC-MS analysis.
The cleaved peptide was applied to a C18 column and attached to an Easy nLC 1200 system (Thermo Fisher Scientific, USA) using 12ml of 100% solution a (0.1% formic acid) for on-line desalting. The peptides were separated on a C18 (prosil-pur C18-aq 1.9 μm, china) capillary column (150 μm. Times.250 mm). The gradient is that 8-12% of phase B (80% ACN,0.1% formic acid) acts for 5min,12-30% of phase B acts for 30min,30-40% of phase B acts for 9min,40-95% of phase B acts for 1min,95% of phase B maintains for 15min, and the flow rate is 600nL/min. The eluted polypeptides were analyzed by Q-exact HF-X (Thermo Fisher Scientific, USA) with the source parameters set to spray voltage 2.1kv; the temperature of the ion transfer tube is 320 ℃; s lens,60. All data acquisitions were positive ion scanned.
And the database building data acquisition adopts a data dependent acquisition mode (DDA) to acquire data within the range of 350-1500 m/z. The resolution parameter was set to 120,000,agc value to 3e6. The first 40 high signal intensity ions fragment preferentially. The resolution of the obtained mass spectral parameters was 15000, the AGC value was 5e4, the isolation window was 1.6Da, the NCE was 27, the maximum IT time was 45MS, and the dynamic exclusion time was 16s.
Clinical sample data is acquired using a data independent acquisition mode (DIA). Full mass scanning was performed in the range of 350-1500 m/z, and the following parameters were obtained, resolution 60000 and agc value 3e6. Adopt toDIA mass spectra were acquired with 42 acquisition windows ranging from 350 to 1500m/z, resolution 30000, AGC 1 × 10 6 The step NCE is 25.5, 27, and 30, respectively.
All DIA original files of Q-active HF-X are processed using DIA-nn (version 1.81). Direct DIA experimental analysis procedures were applied to UniProt human database (2022.03.17) with cysteine iodoacetamidomethyl (C) as the fixed modification and oxidation (M) and acetylation (protein N-term) as the variable modifications. Trypsin miscut settings were 2 sites, 1% protein and peptide errors of FDR. Protein databases were built using fragpipe (v 18.0) software.
A series of experiments were designed to evaluate the effectiveness of this strategy, including: 1. comparing the qualitative depth and the reproducibility of DMB bead enrichment with those of the traditional enrichment method; 2. comparing different incubation times and enzyme digestion methods of the plasma and the DMB beads; 3. the quantification of the gradient proteins in plasma was observed from the characteristics of the protein corona components obtained in different volumes of plasma samples and DMB bead incubations. Serial evaluation experiments were searched directly using DIA-NN software.
Finally, plasma samples of 36 MSA disease groups and healthy controls were analyzed. The pooled plasma proteins were fractionated into 18 fractions with alkaline reverse phase for DDA database establishment. DIA-NN was analyzed qualitatively and quantitatively by building a database. The obtained regulatory protein is further subjected to ELISA quantitative verification in the same batch of plasma.
The same plasma was processed for high-abundance protein removal (2 and 14 high-abundance protein removals), hexapeptide ligand enrichment, and DMB bead enrichment. Comparing the identification result with the pretreatment result without removing high abundance, it can be seen from fig. 2 that the DMB bead method can significantly increase the identification number of plasma proteins to nearly 2000, and the unique identification number reaches 1031. KEGG pathway analysis of these unique proteins suggests that many proteins are involved in brain disease-related pathways, including alzheimer's disease, parkinson's disease, and neuronal generation pathways, providing more observation variables for brain disease studies. Comparing the results of the identification data with the human protein database information, FIG. 3 shows that the DMB beads have a far superior enrichment capacity for proteins at concentrations in the μ g/L and ng/L ranges than other processes.
Fig. 4 shows that DMB beads are enriched to a lower abundance protein concentration more than one order of magnitude lower than other methods, and are enriched to more low abundance proteins than other methods.
Plasma was freshly collected from 3 different healthy volunteers (2 females, 1 male), mass spectrometry was performed after parallel enrichment of DMB beads for 3 times, and qualitative and quantitative reproducibility of plasma protein enrichment was evaluated. The results showed that a total of 2059 proteins were identified between different individuals, accounting for more than 83% of the amount of protein identified in each sample. In addition, quantitative reproducibility of DMB bead enrichment was also analyzed. In 3 replicate enrichment experiments of the same sample, proteins with CV <20% accounted for more than 87% of all proteins.
Time-efficiency is a matter that must be considered for clinical sample analysis. To reduce the processing time and improve the sample throughput, the effect of DMB bead and plasma co-incubation time on the enrichment results was examined. Quantitative data for different incubation times (0.5 h, 1h, 2h and 4 h) had a good correlation coefficient (r > 0.9) at any two time points, while the amount of protein identified did not increase significantly with increasing incubation time. Unlike traditional eluent digestion methods, DMB beads allow proteins to be adsorbed onto the beads for subsequent reduction, alkylation, and digestion, thereby saving elution and tube transfer time. The final results show that direct enzymatic hydrolysis on beads effectively increased the number of protein identifications and reduced protein losses (fig. 5).
The rate of e.coli protein quantification and changes in plasma protein were detected by adding graded amounts of e.coli protein to equal amounts of plasma. The results are shown in FIGS. 6 and 7, in which the plasma protein fluctuates less in the ratio change of 0.94 to 1.25; coli protein exhibited a gradient in ratio, although there was a ratio compression in LC-MS detection. This experiment suggests that the enrichment of magnetic material is related to the abundance of proteins in the sample, rather than a completely disordered enrichment state. Quantitative comparison of low abundance proteins in the sample can be performed after DMB bead enrichment.
36 plasma samples were analyzed using the established protocol of the assay. The difference between the MSA group and the HC group in terms of age, body Mass Index (BMI), blood routine index and the like has no statistical significance (p > 0.05), and through differential proteomics analysis, 215 proteins are up-regulated and 184 proteins are down-regulated (p < 0.05). Using these differential proteins, the MSA group can be distinguished from the healthy control group. The functional classification of the differential proteins was further analyzed. The up-regulated protein function was found to be concentrated on protein phosphorylation, antibacterial peptide and peptidase activities and immune responses, consistent with plasma transcriptomic data. Downregulation of proteins is associated with the maintenance of actin and blood brain barrier function. This is related to the phenotypic characteristics exhibited by the disease. Among these differential proteins, immune-related proteins and brain disease-related proteins are the focus of research. These proteins are present in very low plasma concentrations, usually detectable only in cerebrospinal fluid, and can now be detected and quantified by enrichment with magnetic material.
In the invention, the qualitative and quantitative depth and analysis efficiency of plasma proteomics are greatly improved under the promotion of a depth mining strategy.
Comparative example 1
The detection of proteins in the sample was performed according to the method of example 1, except that DMB beads were not used, but consumables reported in the literature were used, including: si-NH2/SiO2-OH/TiO2/SiO2N + -CH 2-SO 3- (prepared by reference to CN 112710755A); SP-003/SP-007/SP-011 (prepared by reference U.S. Pat. No. 2,5046/0215685); MMN-NHCH3/-SO3H/-Ti4+/COOH (cf. Yuanyuun Liu et al, synthesis of Surface-Functionalized Molybdenum substrates for Efficient Adsorption and Deep Profiling of the Human plasmid by Data-Independent acquisition. Anal Chem.2022Nov 1 (43): 14956-14964 preparation); IONPs (prepared with reference to CN 114791466A).
The results are shown in FIG. 8.
Through comparison between example 1 and comparative example 1, it can be found that the identification result of the material used in the method can be more than doubled compared with other materials, and the method shows the advantages of the material in protein enrichment.
In the present invention, biomarker studies were performed on a variety of systemic wasting diseases using a DMB bead (magnetic covalent organic framework nanomaterial) enrichment-online enzymatic digestion-DIA detection-DIA-nn library search strategy. This strategy indicates that the depth of plasma protein study is increased by more than 3-fold in protein amount compared to other commercial consumables. Qualitative and quantitative analysis was performed on a conventional 2000+ protein/sample. Qualitative and quantitative stability of the sample enrichment assay was demonstrated, with a quantitative CV of less than 20% for over 87% of the proteins. The enrichment, enzymolysis and desalination based on DMB beads are an efficient analysis process, so that the time can be saved, and the loss can be reduced. In the research of MSA disease markers, 18 differential proteins which are related to nervous system diseases or specifically expressed in brain tissues are identified, and the feasibility and superiority of the method for the research of clinical biomarkers are proved.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (10)
1. A method for detecting a protein in a sample, the method comprising the steps of:
s1, mixing the sample with magnetic nanoparticles to obtain protein-bound magnetic nanoparticles;
s2, sequentially carrying out reduction treatment, alkylation treatment, washing treatment and enzyme digestion treatment on the protein-bound magnetic nanoparticles to obtain an enzyme digestion product;
s3, performing mass spectrum detection on the enzyme digestion product;
the magnetic nanoparticles comprise ferroferric oxide nanoparticles and a covalent organic framework formed on the ferroferric oxide nanoparticles, wherein the covalent organic framework is obtained by reacting 1,3, 5-trimethylresorcinol and 1, 4-diaminobenzene.
2. The method of claim 1, wherein the magnetic nanoparticles have a particle size of 0.1 to 0.3 μm.
3. The method according to claim 1 or 2, wherein the content of the covalent organic framework in the magnetic nanoparticles is 2-4 parts by weight per part by weight of the ferroferric oxide nanoparticles.
4. The method of claim 1, further comprising preparing the magnetic nanoparticles by:
SS1, dissolving ferric trichloride hexahydrate and sodium acetate in ethylene glycol to obtain a homogeneous transparent solution; the weight ratio of ferric trichloride hexahydrate to sodium acetate is 1; the dosage of the glycol is 15-25mL relative to 1g of ferric trichloride;
SS2, carrying out a first solvothermal reaction on the homogeneous transparent solution at the temperature of 150-250 ℃ for 4-24 hours to obtain black powder;
SS3, mixing the black powder, 1,3, 5-trimethylresorcinol and 1, 4-diaminobenzene in a weight ratio of 1:1-2:1-2 in ethanol and carrying out a second solvothermal reaction at 150-250 ℃ for 20-80 hours; the amount of ethanol used is 15-25mL relative to 1g of the black powder.
5. The method of claim 1, wherein the sample is whole blood, plasma, serum, or cerebrospinal fluid.
6. The method of claim 5, wherein the sample is mixed with the magnetic nanoparticles after 1-3 fold dilution.
7. The method of claim 6, wherein the amount of the magnetic nanoparticles is 0.05-0.1mg relative to 1mL of the diluted sample.
8. The method of claim 1, wherein the reducing treatment conditions comprise: the reducing agent is tris (2-carboxyethyl) phosphine, the concentration of the reducing agent is 0.01-0.02 mu M, the time is 10-30 minutes, and the temperature is 92-98 ℃; the alkylation conditions include: the alkylating reagent is iodoacetamide, the concentration of the alkylating reagent is 0.025-0.050 mu M, the time is 10-30 minutes, and the temperature is 10-30 ℃.
9. The method according to claim 1, wherein the washing treatment comprises washing with an acetonitrile solution containing 70 to 90 vol% of acetonitrile in water; the enzymatic treatment comprises enzymatic cleavage using trypsin.
10. The method of claim 1, wherein subjecting the cleaved product to mass spectrometric detection comprises qualitative and/or quantitative detection.
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