阅读说明：本技术 一种非标记蛋白质组学检测方法及装置 (Non-labeled proteomics detection method and device ) 是由 丁显廷 王丽萍 朱大为 于 2021-11-23 设计创作，主要内容包括：本发明公开了一种痕量细胞在线分离串联质谱检测的非标记蛋白质组学方法,涉及单细胞蛋白质组学检测领域,由微流控系统实现细胞分离、样品制备、以及串联质谱检测。本发明通过建立微流控系统实现细胞的精准计数和微量生物样本的前处理,从而实现对单个细胞蛋白信息的精确定量；进一步开展CTCs高通量蛋白质组学的检测,利用CTCs的蛋白质组学信息监测个体对化疗的耐药反应,对进一步指导个体化用药具有重大的意义。(The invention discloses a non-labeled proteomics method for trace cell online separation tandem mass spectrometry detection, relates to the field of single cell proteomics detection, and realizes cell separation, sample preparation and tandem mass spectrometry detection by a microfluidic system. According to the invention, the accurate counting of cells and the pretreatment of a trace biological sample are realized by establishing a microfluidic system, so that the accurate quantification of single cell protein information is realized; the detection of CTCs high-throughput proteomics is further developed, the proteomics information of CTCs is used for monitoring the drug resistance reaction of an individual to chemotherapy, and the method has great significance for further guiding individualized medication.)

1. A method for unlabeled proteomic detection, comprising the steps of:

step 1, preparing a chip;

step 2, separating cells and counting through a microfluidic system;

step 3, preparing a proteomics sample by microfluidics;

and 4, collecting DDA data by the 4D mobility mass spectrum.

2. The unlabeled proteomics detection method of claim 1, wherein the step 1 specifically comprises the steps of:

step 1.1, pouring PDMS and a curing agent into a culture dish on the surface of a silicon wafer according to the proportion of 11:1, and baking for 45 minutes at 85 ℃;

step 1.2, adhering the nut to the PDMS solidified surface near the middle of the semicircular channel for 50 seconds by using oxygen plasma;

step 1.3, pouring the mixture of the PDMS obtained in the step 1.1 and the curing agent into a culture dish until a nut is covered, and baking for 1 hour;

step 1.4, stripping PDMS with nuts from a dish, and adhering the PDMS to a glass substrate after oxygen plasma treatment for 50 s;

step 1.5, punching holes at an inlet and an outlet, wherein the inlet and the inner outlet adopt punching machines with the diameter of 0.8mm, and the outer outlet adopts a punching machine with the diameter of 2.5 mm;

step 1.6, sticking a 20-micron filter membrane on the top of the outer outlet;

step 1.7 at room temperature, the chip was blocked with 1% BSA for 1h, washed, and used in a 37 ℃ ovenThe pipe is connected with the steel pipe, and is inserted into the inlet and the inner outlet for fluid injection and collection.

3. The unlabeled proteomics detection method of claim 1, wherein step 3 comprises the steps of:

step 3.1, preparing target cells, diluting, adding a microfluidic system through an inlet, removing impurities through a cell channel, and entering a sample cell;

step 3.2, washing the chip, shooting a cavity image through a laser confocal microscope, and counting cells;

3.3, removing the filter membrane, screwing the bolt into the nut, and fixing the cells in the outer outlet cavity;

step 3.4, placing the chip in an oven at 80 ℃ for 30 minutes;

step 3.5, adding 5 mu L of lysate into an external outlet, sealing, and culturing at 60 ℃ for 1 h;

step 3.6, adding 5 mu L of trypsin from an inner outlet, sealing, incubating overnight at 37 ℃, and collecting a sample from an outer outlet;

step 3.7, adding 1% formic acid to terminate the reaction, and spin-drying;

and 3.8, adding formic acid water for redissolving, and performing liquid mass analysis.

4. The non-labeled proteomic detection method of claim 2 or 3, wherein 50mM NH is used in step 1.7 and step 3.2₄HCO₃The solution washes the chip.

5. The method for the non-labeled proteomic detection of claim 3, wherein the lysate is 0.1-0.5% DDM, 1-5mM TCEP, 2-10mM CAA in 50mM NH₄HCO₃And (4) preparing.

6. The method for unlabeled proteomic detection of claim 5, wherein the lysate is 0.1% DDM, 1mM TCEP, 2mM CAA.

7. The method for unlabeled proteomic detection of claim 3, wherein the mass ratio of trypsin to protein to be detected is 1: 10.

8. The unlabeled proteomics detection method of claim 1, wherein step 4 comprises the steps of:

step 4.1 the liquid system is serial nanoElute time TOF Pro, mobile phase A is 0.1% formic acid water, mobile phase B is 0.1% formic acid acetonitrile, 40min gradient is as follows: 5-24% B25 min, 24-36% B5 min, 36-80% B2 min, 80-80% B8 min;

step 4.2 resuspend the peptide fragment 5. mu.L, load 4. mu.L of micro trap cartridge chromoXP C18CL at a maximum pressure of 275bar, separate the peptide fragment using analytical column C18;

step 4.3, adopting positive ion DDA parallel accumulation sequence fragmentation mode for timsTOF Pro, setting the capillary voltage to be 1400V, the range of primary and secondary mass-to-charge ratios to be 300 m/z-1500 m/z, and the range of ion mobility to be 1/K0 to be 0.60Vs/cm²～1.60Vs/cm²。

9. The utility model provides a micro-fluidic chip is detected to non-mark proteomics, its characterized in that contains cell channel (5) and sample cell, and cell channel (5) both ends set up import (52) and interior export (53), and the sample cell includes outer export, nut (3) and (7), bolt (1), cell channel (5) pass through semicircle type passageway with the sample cell and are connected, and entry (52) and interior export (53) adopt the piercing press that the diameter is 0.8-1.0mm, and the outer export adopts the piercing press that the diameter is 2-3mm, semicircle type passageway inner radius is 6-6.5mm, and external diameter 6.8-7.0 mm.

10. The unlabeled proteomic detection microfluidic chip of claim 9, wherein the outlet outside the sample cell is covered with a 20 μm pore size filter.

Technical Field

The invention relates to a non-labeled proteomics method for trace cell online separation tandem mass spectrometry detection, in particular to a non-labeled proteomics detection method for realizing cell separation, sample preparation and tandem mass spectrometry detection through a microfluidic system.

Background

In proteomics research, except for sensitivity and high resolution influencing factors of mass spectrometer hardware, sample pretreatment, mass spectrum data acquisition and data analysis all influence proteomics research.

The sample pretreatment process is complex and needs to be carried out according to the concrete requirementsThe experimental objective of (a) was optimized to reduce the degradation and modification of proteins during the sample processing phase, releasing as many peptide fragments as possible for the final mass spectrometric detection. Wherein, the proteomics detection of the micro-sample is a technical bottleneck, and the non-labeled proteomics detection can not be carried out by using the traditional method for samples such as flow sorting cells, embryo culture, laser microdissection, Circulating Tumor Cells (CTCs), fine needle puncture and the like. Conventional proteomics studies are based on the study of cell populations, averaging a large amount of cell information, and generally require that the number of cells be at least greater than 10⁶. Whereas micro-sample proteomics studies can address cellular heterogeneity. The traditional proteomics sample preparation has the problems of large system and tube adsorption at present.

The traditional mass spectrometry technology is influenced by low acquisition speed, and the identification depth of a single sample is difficult to improve; post-translational modification plays an important role in the life process, but because the proportion of modified polypeptides is generally low, the types of modified polypeptides are more, the number of modified position isomers is more, and the modified polypeptides are limited by the sensitivity and the separation capability of the traditional mass spectrometry technology, the identification depth of the modified polypeptides is usually insufficient, and the modified position isomeric polypeptides cannot be accurately identified and quantified.

Although the proteomics mass spectrometry technology is mature at present and has great improvements in three aspects of proteomics micro sample pretreatment, liquid phase and mass spectrometry and data acquisition mode, numerous challenges still face. Improved methods based on unlabeled proteomics require a larger sample size and research based on TMT labeled proteomics is more costly. The micro-fluidic platform is adopted to obtain a good identification result for proteomics of a trace sample, the demand for cells is greatly reduced, the detection of the proteomics of less than 100 cells can be realized, but the design is relatively complex and is not easy to be adopted by other mass spectrometry laboratories, and meanwhile, after the sample preparation of the micro-fluidic platform is finished, the high requirement for the operation of a liquid quality machine is also provided by the direct sample injection of a chromatographic column, a mature chromatographic column preparation system is required, meanwhile, the time for running one sample is relatively long, and the continuous automatic sample injection is not available.

Therefore, those skilled in the art are devoted to develop a proteomics detection method for realizing accurate cell counting and pretreatment of micro biological samples, thereby realizing accurate quantification of single cell protein information

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is to achieve qualitative and relative quantification of single-cell protein information.

In order to achieve the aim, the invention provides a non-labeled proteomics method for trace cell online separation tandem mass spectrometry detection.

The method comprises the following steps:

step 1, chip preparation

Step 1.1 pouring Polydimethylsiloxane (PDMS) and a curing agent (Sylgard 184, Dow Corning) into a culture dish on the surface of a silicon wafer according to the proportion of 11:1, and baking for 45 minutes at 85 ℃;

step 1.2, adhering the nut to the PDMS solidified surface near the middle of the semicircular channel for 50 seconds by using oxygen plasma;

step 1.3, mixing PDMS and a curing agent, pouring the mixture into a culture dish until the nut is covered, and baking for 1 hour.

Step 1.4, stripping PDMS with nuts from a dish, and adhering the PDMS to a glass substrate after oxygen plasma treatment for 50 s;

and 1.5, punching at an inlet and an outlet. The inlet and the inner outlet adopt punching machines with the diameter of 0.8mm, and the outer outlet adopts a punching machine with the diameter of 2.5 mm;

step 1.6, sticking a 20-micron filter membrane on the top of the outer outlet;

step 1.7 the chip was treated with 1% BSA at room temperature for 1h to block the chip surface and prevent unspecified proteins from binding. With 50mM NH₄HCO₃Bovine serum albumin was removed by washing and used in a 37 ℃ oven. FlexibilityThe pipe is connected with the steel pipe and inserted into the inlet and the inner partAnd an outlet for fluid injection and collection.

Step 2, cell separation and counting by microfluidic system

Preferably, 50mM NH is used₄HCO₃Washing the chip to replace PBS, and taking a cavity image through a Zeiss LSM 880 and Germany microscope for accurate cell counting;

step 3, preparation of proteomics samples by microfluidics

Step 3.1, preparing target cells, diluting, adding the target cells into a microfluidic system through a sample introduction end, removing impurities through a cell channel, and allowing the target cells to enter a sample cell;

step 3.2 with 50mM NH₄HCO₃Washing the chip to replace PBS, and taking a cavity image through a Zeiss LSM 880 and Germany microscope for accurate cell counting;

step 3.3, the filter membrane is removed;

step 3.4, screwing the bolt into the nut to fix the cell in the outer outlet cavity;

step 3.5, placing the chip in an oven at 80 ℃ for 30 minutes to denature cell protein;

step 3.6 Add 5. mu.L (0.1% DDM, 1mM TCEP, 2mM CAA in 50mM NH4HCO3) to the external outlet;

and 3.7, sealing an outlet of the culture medium by using a sample injection bottle sealing gasket and an electric adhesive tape, and culturing for 1h at the temperature of 60 ℃.

Step 3.8 then add 5 μ L trypsin (total trypsin (w): protein (w): 1:10) from the internal outlet, seal with a sample vial gasket and electric tape, incubate overnight at 37 ℃, and finally, take the sample from the external outlet;

step 3.9, adding 1% formic acid to terminate the reaction, and spin-drying;

step 3.10, adding formic acid water for redissolving, and carrying out liquid mass analysis;

step 4, carrying out DDA data acquisition by 4D mobility mass spectrum

Step 4.1 the liquid system is serial nanoElute time TOF Pro, mobile phase A is 0.1% formic acid water, mobile phase B is 0.1% formic acid acetonitrile, the liquid phase gradient is as follows: the 40min gradient was as follows: 5-24% B (25min), 24-36% B (5min), 36-80% B (2min), 80-80% B (8 min);

step 4.2 resuspending the peptide fragment by 5. mu.L, and taking 4. mu.L to load it onto a micro trap cartridge chromoXP C18CL (5. mu.m) at a maximum pressure of 275barAB SCIEX), peptide fragment separation using analytical columns (C18, 75. mu. m.times.25 cm, 1.7. mu.m, IonOpticks, Australia-Aurora);

step 4.3timsTOF Pro employs positive ion DDA parallel accumulation sequence fragmentation (PASEF) mode. The capillary voltage was set at 1400V. The first-level and second-level mass-to-charge ratios are 300m/z to 1500m/z, and the ion mobility range (1/K0) is 0.60Vs/cm2 to 1.60Vs/cm 2.

The invention also provides a non-labeled proteomic detection microfluidic chip which comprises a cell channel 5 and a sample cell, wherein an inlet 52 and an inner outlet 53 are arranged at two ends of the cell channel 5, the sample cell comprises an outer outlet, nuts 3 and 7 and a bolt 1, the cell channel 5 is connected with the sample cell through a semicircular channel, the inlet 52 and the inner outlet 53 adopt a punching machine with the diameter of 0.8-1.0mm, the outer outlet adopts a punching machine with the diameter of 2-3mm, and the inner radius of the semicircular channel is 6-6.5mm and the outer diameter is 6.8-7.0 mm.

Preferably, the outlet (3) of the sample cell is covered with a filter membrane with a pore size of 20 μm.

In addition, the invention also provides application of the non-labeled proteomics detection method in CTC cell high-throughput proteomics detection.

The invention has the advantages that:

1. accurate counting of cells and pretreatment of a trace biological sample are realized by establishing a microfluidic system, so that accurate quantification of single cell protein information is realized, and compared with the traditional pretreatment of a proteomic sample, the operation is simpler and more convenient, and compared with a labeled proteomic detection method, the cost is greatly reduced;

2. the established microfluidic system is combined with a 4D mobility mass spectrum to carry out DDA data acquisition method, the detection of CTCs high-throughput proteomics is further carried out, and the proteomics information of the CTCs is used for monitoring the drug resistance response of an individual to chemotherapy.

Drawings

FIG. 1 is a schematic diagram of a chip structure according to the present invention;

FIG. 2 is a graph of the effect of different loading on the mean identification of proteomes for quantifiable protein combinations;

FIG. 3 is the effect of DDA, DIA and dDIA on the identifiable group of proteins;

FIG. 4 is the number of quantifiable proteomes for different cell numbers and the number of intersections;

FIG. 5 shows the identification of different MCF7 and CTCs proteomes isolated by microfluidic systems (A-C:106, 119 and 107 MCF7 cells, D-F:57, 68 and 58 MCF7 cells, G-H:7 and 5 CTCs).

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

In the drawings, structurally identical elements are represented by like reference numerals, and structurally or functionally similar elements are represented by like reference numerals throughout the several views. The size and thickness of each component shown in the drawings are arbitrarily illustrated, and the present invention is not limited to the size and thickness of each component. The thickness of the components may be exaggerated where appropriate in the figures to improve clarity.

Example 1 preparation of microfluidic System

(1) PDMS and a curing agent (Sylgard 184, Dow Corning) were poured into a petri dish on the surface of a silicon wafer at a ratio of 11:1 and baked at 85 ℃ for 45 minutes.

(2) The nut was bonded to the PDMS setting surface near the middle of the semicircular channel with oxygen plasma for 50 seconds.

(3) PDMS and curing agent mixed into the culture dish, until covering the nut, baking for 1 hours.

(4) The PDMS with the nut was peeled off the dish, oxygen plasma treated for 50s, and then adhered to a glass substrate.

(5) And holes are punched at the inlet and the outlet. The inlet and the inner outlet adopt punching machines with the diameter of 0.8mm, and the outer outlet adopts a punching machine with the diameter of 2.5 mm.

(6) The top of the outer outlet is stuck with a filter membrane with the aperture of 20 mu m.

(7) The chip was treated with 1% BSA at room temperature for 1h to block the chip surface and prevent unspecified proteins from binding. With 50mM NH₄HCO₃Bovine serum albumin was removed by washing and used in a 37 ℃ oven. FlexibilityThe pipe is connected with the steel pipe, and is inserted into the inlet and the inner outlet for fluid injection and collection.

EXAMPLE 2293 preparation of a proteome sample of T cells

(1) Preparing target cells, diluting, adding the diluted target cells into a microfluidic system through a sample introduction end, and introducing the diluted target cells into a sample cell.

(2) With 50mM NH₄HCO₃The chip was washed, PBS replaced, and chamber images were taken by Zeiss LSM 880, german microscope for accurate cell counting.

(3) The filter membrane is removed.

(4) The bolt is screwed into the nut, securing the cell in the outer exit chamber.

(5) The chip was placed in an oven at 80 ℃ for 30 minutes to denature the cellular proteins.

(6) mu.L (0.1% DDM, 1mM TCEP, 2mM CAA in 50mM NH)₄HCO₃) Adding to the outer outlet.

(7) Sealing the outer outlet with a sample bottle gasket and an electric adhesive tape, and culturing at 60 deg.C for 1 h.

(8) Then 5 μ L of trypsin (total trypsin (w): protein (w): 1:10) was added from the internal outlet, sealed with a sample vial gasket and electric tape, and incubated overnight at 37 ℃. Finally, samples were taken from the external outlet.

(9) Adding 1% formic acid to terminate the reaction, and spin-drying;

(10) adding formic acid water for redissolution, and carrying out liquid mass analysis.

Example 3 isolation, enumeration and proteome sample preparation of MCF7 cells

(1) MCF7 cell concentration 5X 10⁴cells/mL and 1X 10³cells/mL and 5X 10, respectively⁴cells/mL WBCs are mixed, and the mixed solution is added into a microfluidic system for 1mL/h through a sample introduction end and enters a sample cell.

(2) Smaller cells and leukocytes flow out of the internal outlet, and MCF7 cells flow out of the external outlet through the filter membrane.

(3) With 50mM NH₄HCO₃The chip was washed, PBS replaced, and chamber images were taken by Zeiss LSM 880, german microscope for accurate cell counting.

(4) The filter membrane is removed.

(5) The bolt is screwed into the nut, securing the cell in the outer exit chamber.

(6) The chip was placed in an oven at 80 ℃ for 30 minutes to denature the cellular proteins.

(7) mu.L (0.1% DDM, 1mM TCEP, 2mM CAA in 50mM NH4HCO3) was added to the external outlet.

(8) Sealing the outer outlet with a sample bottle gasket and an electric adhesive tape, and culturing at 60 deg.C for 1 h.

(9) Then 5 μ L of trypsin (total trypsin (w): protein (w): 1:10) was added from the internal outlet, sealed with a sample vial gasket and electric tape, and incubated overnight at 37 ℃. Finally, samples were taken from the external outlet.

(10) Adding 1% formic acid to terminate the reaction, and spin-drying;

(11) adding formic acid water for redissolution, and carrying out liquid mass analysis.

Example 4 isolation, enumeration and proteome sample preparation of CTC cells

(1) Blood of a clinical patient is subjected to erythrocyte lysis, and the blood enters a sample cell after being added into a microflow system for 1mL/h through a sample introduction end.

(2) Smaller cells and leukocytes flow out of the internal outlet, and CTC cells flow out of the external outlet through the filter.

(3) With 50mM NH₄HCO₃The chip was washed, PBS replaced, and chamber images were taken by Zeiss LSM 880, german microscope for accurate cell counting.

(4) The filter membrane is removed.

(5) The bolt is screwed into the nut, securing the cell in the outer exit chamber.

(6) The chip was placed in an oven at 80 ℃ for 30 minutes to denature the cellular proteins.

(7) mu.L (0.1% DDM, 1mM TCEP, 2mM CAA in 50mM NH)₄HCO₃) Adding to the outer outlet.

(8) Sealing the outer outlet with a sample bottle gasket and an electric adhesive tape, and culturing at 60 deg.C for 1 h.

(9) Then 5 μ L of trypsin (total trypsin (w): protein (w): 1:10) was added from the internal outlet, sealed with a sample vial gasket and electric tape, and incubated overnight at 37 ℃. Finally, samples were taken from the external outlet.

(10) Adding 1% formic acid to terminate the reaction, and spin-drying;

(11) adding formic acid water for redissolution, and carrying out liquid mass analysis.

The liquid mass analysis procedure was referred to in summary step 4.

While the preferred embodiments of the present invention have been illustrated and described in detail, it should be understood that modifications and variations can be made by persons skilled in the art without the use of inventive faculty, and in light of the above teachings. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.