WO2022117115A1 - On-line automatic analysis device and analysis method for phosphoproteomics - Google Patents

Abstract

An on-line automatic analysis device and an analysis method for phosphoproteomics. The device is a liquid chromatography-mass spectrometer, and the liquid chromatography comprises a phosphorylated peptide capture column (4) and an analysis column (8), and the phosphorylated peptide capture column (4) is an ATP-modified immobilized metal ion affinity chromatographic column. The ATP-modified immobilized metal ion affinity chromatographic column is obtained by mixing and stirring potassium water glass, γ-glycidyl ether oxypropyl trimethoxy silane and water-dissolved disodium adenosine triphosphate, and then adding water-dissolved formamide and stirring same to obtain a reaction solution, filling up a chromatographic column with the reaction solution, then reacting and curing the reaction solution in the filled chromatographic column, and then washing same. The primary advantage is that researchers are released from complicated manual operations such as loading metal ions, loading samples, washing, eluting, desalting, volatilizing and redissolving, which are required to enrich phosphorylated peptides, such that the experimental efficiency is improved.

Description

An online automated analysis device and analysis method for phosphorylation proteomics technical field

The invention belongs to the fields of chromatography and mass spectrometry, and in particular relates to an online automated analysis device and an analysis method for phosphorylated proteomics for automated online analysis of phosphorylated polypeptides on a nano-liter liquid chromatography-mass spectrometer.

Background technique

Many important life regulation processes are related to protein phosphorylation. Qualitative and quantitative analysis of protein phosphorylation events in cells is helpful to reveal the regulatory mechanism of phosphorylation-modified proteins in signaling pathways, to mine key genes or to guide drug development. target. Liquid chromatography-high resolution mass spectrometry combined with bottom-up analysis strategy is the core method of current proteomics research. However, due to the extremely low abundance of phosphorylated peptides, poor ionization efficiency and signal inhibition by non-phosphorylated peptides, This makes the direct analysis of phosphopeptides from whole protease digests very difficult, and selective enrichment of phosphopeptides from digested samples before analysis is an essential operation.

Immobilized metal ion affinity chromatography (IMAC) is the most important enrichment technique in phosphoproteomics, and chelating ligands such as iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) can convert metal cations. (such as Cu ²⁺ , Fe ³⁺ , Ni ²⁺ or Ga ³⁺ , etc.) are immobilized on the surface of the solid-phase matrix, and these immobilized metal ions can be combined with the phosphate group of the polypeptide through Lewis acid-base interaction, so as to form a complete The protease digest is enriched with phosphorylated peptides, which are then eluted by alkaline buffers, and then subjected to steps such as acidification, desalting, and concentration before mass spectrometry analysis. Since IMAC is usually in the form of microspheres, magnetic materials or microcolumns, the above process needs to be performed manually by eddy current, centrifugation/magnetic suction or pipette suction, which is time-consuming and labor-intensive, and the tedious manual operation will inevitably lead to cause large errors or errors.

An idea to solve the above problems is to invent an IMAC in the form of a chromatographic column, which can be combined with a liquid chromatography system. It can realize automatic enrichment and analysis of phosphorylated peptides. Nano-liter liquid chromatography-mass spectrometry is used in practical proteomics analysis tasks, and the development of nano-liter phosphorylated peptide enrichment columns often encounters high back pressure, low sample loading rate, and poor sensitivity. Another major difficulty is that the elution of phosphorylated peptides from the capture column requires a high pH solution, which is incompatible with the loading conditions of the subsequent C18 analytical column. Finally, how to design the connection of the liquid phase system and the automated analysis program are also technical difficulties. , so there is no commercial device available for online and automated analysis of phosphorylated proteomes.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an online automated analysis device for phosphorylation proteomics.

The online automated analysis device for phosphorylation proteomics of the present invention is a liquid chromatography-mass spectrometry instrument, and the liquid chromatography includes a phosphorylated peptide capture column and an analysis column, characterized in that the Phosphorylated peptide capture column is an ATP-modified immobilized metal ion affinity chromatography column;

The preparation method of the ATP-modified immobilized metal ion affinity chromatographic column is:

Mix and stir potassium water glass, γ-glycidyl etheroxypropyltrimethoxysilane and water-dissolved adenosine triphosphate disodium, then add water-dissolved formamide and stir to obtain a reaction solution, fill the column with the reaction solution, and then fill The reaction solution of the chromatographic column is reacted and solidified, and then washed to obtain an ATP-modified immobilized metal ion affinity chromatographic column.

The schematic diagram of the preparation principle of the ATP-modified immobilized metal ion affinity chromatography column of the present invention is as follows:

1. Preparation of potassium water glass

mKOH+nSiO ₂ →mK ₂ O·(nm)SiO ₂ +mH ₂ O

2. Hydrolysis of potassium water glass under formamide and heating conditions

3. The reaction of silicon coupling agent and ATPNa ₂ carried out simultaneously with step 2:

4. ATP modification on silica substrate

5. Filler plays an enrichment function

Preferably, the dosage ratio of the potassium water glass, γ-glycidyl ether oxypropyltrimethoxysilane, adenosine triphosphate disodium salt and formamide is 500-2000:1-10:2-50:20-130.

Further preferably, the amount-to-mass ratio of the potassium water glass, γ-glycidyl ether oxypropyltrimethoxysilane, adenosine triphosphate disodium salt and formamide is 1000:6:7.5:68.

Preferably, the modulus of the potassium water glass is in the range of 2-4, and the Baumé degree is in the range of 20-50.

Further preferably, the modulus of the potassium water glass is 3.3, and the Baumé degree is 40.

Preferably, the curing is curing at a temperature of 100° C. for 10 hours; the washing is washing with 1M ammonium nitrate, 0.1M nitric acid and water successively.

Preferably, the chromatographic column is an elastic quartz capillary.

Further preferably, the elastic quartz capillary is an elastic quartz capillary with an outer diameter of 360 microns, an inner diameter of 150 microns and a length of 15 cm

Preferably, the online automated analysis device for phosphorylation proteomics includes a sample loading part and an analysis part, and the sample loading part includes a first group of mobile phase storage bottles and a second group of mobile phase storage bottles , degasser, sample pump, autosampler, phosphorylated peptide capture column, the analysis part includes NC pump, C18 pre-column, ten-way valve, C18 analysis column, nano-ESI and high-resolution mass spectrometer, The first group of mobile phase storage bottles, degasser, sample loading pump, six-port valve in the autosampler, phosphorylated peptide capture column and ten-port valve are connected in sequence through pipelines, and the six-port valve is also provided with There is a quantitative loop connected to the two channels of the six-port valve. The autosampler also has a sample tray and a syringe connected to the six-port valve, respectively. The second group of mobile phase storage bottles, NC pump and ten-port valve pass through the pipeline in sequence. There is also a C18 pre-column on the ten-way valve. The C18 pre-column is connected to the ten-way valve through a pipeline, and the ten-way valve is also connected to the C18 analytical column. The outlet end of the C18 analytical column can be connected to the nano-ESI and high-resolution mass spectrometer. Connected in sequence, the ten-way valve is also connected to a waste liquid bottle.

The second object of the present invention is to provide an analytical method for phosphoproteomics, which comprises the following steps:

A. The phosphorylated peptide capture column is eluted with ZrCl ₄ , so that the ATP-modified immobilized metal ion affinity chromatography column packing chelates Zr ⁴⁺ ;

B, take the sample to flow through the phosphorylated peptide capture column of step A to complete the enrichment of phosphorylated peptides;

C, washing liquid flows through the phosphorylated peptide capture column of step B, completes the cleaning to non-specific binding polypeptide;

D. The eluate flows through the phosphorylated peptide capture column of step B, and the enriched phosphorylated peptides are eluted, and the eluted phosphorylated peptides enter the C18 pre-column;

E. The C18 pre-column is eluted with the mobile phase, and the phosphorylated peptide enters the C18 analytical column and is identified by the mass spectrometer.

Preferably:

S1. The injection bottles in the autosampler are respectively filled with 0.1M ZrCl ₄ , sample solution, washing liquid fraction 80% ACN, 1% TFA, eluent 1M NH ₄ H ₂ PO ₄ , washing liquid fraction 40% ACN, 5% _NH4OH ;

S2. The automatic sampler sucks 0.1M ZrCl ₄ into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column under the push of the sample pump → ten-port valve → waste liquid bottle, the mobile phase of the sample pump is the volume fraction 0.1% FA, the mobile phase volume fraction controlled by NC pump is 80% ACN, 0.1% FA flows through the ten-way valve→C18 pre-column→ten-way valve→C18 analytical column;

S3. Dissolve the sample in a solution with a volume fraction of 80% ACN and 1% TFA, the automatic sampler draws the sample solution into the quantitative loop, the six-way valve cuts the valve, and flows through the phosphorylated peptide capture column driven by the sampling pump → ten Pass valve → waste liquid bottle, the mobile phase of the loading pump is 0.1% FA, and the mobile phase controlled by the NC pump is 80% ACN and 0.1% FA flow through the ten-way valve → C18 pre-column → ten-way valve → C18 analysis column;

S4. The automatic sampler draws the washing liquid fraction of 80% ACN and 1% TFA into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column under the push of the sample pump → ten-port valve → waste liquid bottle, upper The mobile phase of the sample pump is 0.1% FA in volume, and the mobile phase controlled by the NC pump with a volume fraction of 0.1% FA flows through the ten-port valve → C18 pre-column → ten-port valve → C18 analytical column;

S5. The ten-port valve cuts the valve, the autosampler draws the eluent 1M NH ₄ H ₂ PO ₄ into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column driven by the sample pump → ten-port valve → C18 pre-column → ten-port valve → waste liquid bottle, the mobile phase of the loading pump is 0.1% FA by volume; the mobile phase controlled by the NC pump with a volume fraction of 0.1% FA flows through the ten-port valve → C18 analytical column; after elution, ten Open the valve and cut the valve, let the mobile phase A of the NC pump with a volume fraction of 0.1% formic acid and 2% acetonitrile flow through the ten-port valve → C18 pre-column → ten-port valve → C18 analytical column, and continue to rinse and remove salt;

S6. The ten-port valve cuts the valve, the automatic sampler absorbs the cleaning liquid fraction of 40% acetonitrile and 5% NH ₄ OH into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column driven by the sampling pump → ten Open valve waste liquid bottle, the mobile phase of the loading pump is 0.1% FA, and the mass spectrometry detection is turned on; the mobile phase volume controlled by the NC pump is 40% ACN, 0.1% FA flows through the ten-way valve → C18 pre-column → ten-way Valve→C18 analytical column→needle→nano ESI source, detected with mass spectrometer.

The filler in the ATP-modified immobilized metal ion affinity chromatography column of the present invention has loose and porous, high specific surface area, low back pressure, good physical and chemical stability, good mechanical stability, high selectivity, high sensitivity, enrichment The advantage of high multiples.

The ATP-modified immobilized metal ion affinity capillary monolithic column prepared by the one-step reaction method has the advantages of simple and rapid preparation steps, high repeatability and yield, and low preparation cost. It can be obtained by only mixing the raw materials uniformly and going through a baking reaction, and has good physical and chemical stability and good reproducibility after repeated use. Compared with the enrichment materials in the form of microspheres or magnetic materials commonly used in the art, the ATP-modified immobilized metal ion affinity capillary monolithic column of the present invention has the potential to be combined with a nanoliter liquid phase system, combined with an automatic sampling system. , which can automate labor-intensive operations such as metal ion loading, sample loading, washing, and elution, and at the same time, the site coverage, detection limit, and selectivity of phosphorylated peptides in the present invention also reach the first-class level in the industry.

Compared with the prior art, the online automated analysis device for phosphorylation proteomics of the present invention has the following beneficial effects:

The present invention provides the design of an on-line analysis device for phosphorylation proteomics, and how to use the device to realize automatic analysis. It is freed from complicated manual operations such as sample loading, washing, elution, desalination, evaporation, and reconstitution, which improves the experimental efficiency. Second, the instrument's precise control of sample injection volume and liquid phase conditions is conducive to reducing manual operations. The resulting errors can improve the consistency of the results of parallel experiments; third, compared with the traditional method of enrichment in centrifuge tubes, the advantage of this combination of trap columns and nanoscale liquid phase systems for online enrichment is that The sample loading efficiency is improved, and the sensitivity is improved a little; finally, the present invention proposes to use 1M NH ₄ H ₂ PO ₄ as the eluent, the acidic NH ₄ H ₂ PO ₄ solution meets the pH requirements of the C18 pre-column, and the phosphoric acid The phosphopeptides can be eluted from the capture column and retained by the subsequent C18 pre-column, and the excess phosphate can be washed away by the loading buffer without affecting the mass spectrometry analysis, which is the key to the realization of this method.

Description of drawings

Figure 1 is a cross-sectional electron microscope image of the ATP-modified immobilized metal ion affinity capillary monolithic column, with dimensions of 200 μm and 5 μm, respectively.

Figure 2 is a schematic diagram of the structure of an online automated analysis device for phosphorylation proteomics, wherein 1. a sample pump; _2. a six-way valve; The sample was dissolved in (by volume, 80% acetonitrile, 1% trichloroacetic acid TFA), 1 mL of washing solution (by volume, 80% acetonitrile, 1% TFA washing solution), 1 mL of 1M ammonium dihydrogen phosphate, 1 mL of wash solution (by volume fraction, 40% acetonitrile, 5% NH ₄ OH), a total of five solutions); 4. Phosphorylated peptide capture column; 5. NC pump; 6. C18 pre-column; 7. Ten-way valve; 8 , C18 analytical column; 9, Nano-ESI ion source; 10, high-resolution mass spectrometer; 11, 20 microliter quantitative loop; 12, waste liquid bottle; 13, sample tray; 14, syringe (sample tray, syringe, six-way Valve and quantitative loop are the four major components of the automatic sampler); 15. The first group of mobile phase storage bottles; 16. The second group of mobile phase storage bottles; 17. Degasser.

3 is a schematic diagram of an automated flow of an online automated analysis device for phosphorylation proteomics;

Figure 4 is a primary mass spectrum of phosphorylated peptides from standard protein α-casein cleavage products by an online automated analyzer for phosphoproteomics.

Figure 5 is the mass spectrogram of the enriched phosphorylated peptides with different loading amounts (from top to bottom: 2ng, 20ng, 40ng, 100ng, 200ng, this amount refers to the mass of the peptides contained in the 10ul injection volume), indicating that The detection limit of the method is as low as 100 fmol.

Figure 6 is a linear relationship between the signal intensity of the phosphorylated peptide and the amount loaded.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be noted that the following embodiments are only for explaining the present invention, and do not limit its content in any form. The design idea of the present invention or the simple substitution of similar substances shall be included within the protection scope of the present invention. Unless otherwise specified, the reagents, materials, methods and equipment used in the present invention are conventional reagents, materials, methods and equipment in the art.

Example 1 Preparation of ATP-modified immobilized metal ion affinity capillary monolithic column

The ATP-modified immobilized metal ion affinity capillary monolithic column prepared by the one-step reaction method specifically comprises the following steps:

S1. Add 740 microliters (about 1000 mg) of potassium water glass (modulus 3.3, Baumé degree 40) to a micro reaction flask, and slowly add 6 microliters (about 6 mg) of γ-glycidyl ether oxypropion under stirring at room temperature trimethoxysilane (GLYMO, Cas number: 2530-83-8), and continued to stir well at room temperature for 30 minutes.

S2. Weigh 7.5 mg of adenosine triphosphate disodium, dissolve it in 160 microliters of deionized water, slowly add it to the reaction solution in step S1, and continue to fully stir at room temperature for 30 minutes.

S3. Take 60 microliters (about 68 mg) of formamide, mix with 40 microliters of deionized water, slowly add it to the reaction solution in step S2, and continue stirring for 1 minute at room temperature to obtain a reaction solution.

S4. Cut out a batch of elastic quartz capillaries with an outer diameter of 360 microns, an inner diameter of 150 microns and a length of 15 cm, insert them into the reaction solution obtained in S3, and fill the capillaries with the reaction solution by capillary phenomenon.

S5. Put the filled capillary into an oven at 100° C. to cure for 10 hours, and cut off 2 cm from both ends of the obtained monolithic column.

S6. Wash the monolithic column with 200 μl each of 1M ammonium nitrate, 0.1M nitric acid and deionized water using a pressure injection cell, thereby obtaining a finished ATP-modified immobilized metal ion affinity capillary monolithic column.

The cross-section of the ATP-modified immobilized metal ion affinity capillary monolithic column is shown in the electron microscope image in Figure 1. Under the field of view of 5 microns, it can be observed that the material is loose and porous, with an average pore size of about 1 micron, which proves that The material has a large specific surface area, which is the basis for its high enrichment efficiency. At the same time, this porous structure can avoid high back pressure during the loading process, and can complete the loading at a higher flow rate.

Example 2

As shown in Figure 2, the online automated analysis device for phosphorylation proteomics in this embodiment includes a sample loading part and an analysis part, and the sample loading part includes a sample loading pump 1, a six-way valve 2, an automatic feeding part Sampler 3 (automatic sampler includes sample tray 13, syringe 14, six-way valve 2 and quantitative loop 11), phosphorylated peptide capture column 4 (that is, the ATP-modified immobilized metal ion affinity capillary monolithic column of Example 1) ), which is mainly responsible for the automated enrichment of phosphorylated peptides. The analysis part includes NC pump 5, C18 pre-column 6, ten-way valve 7, C18 analysis column 8, nano-ESI ion source 9 and high-resolution mass spectrometer 10, and this part is mainly responsible for online analysis of enriched phosphorylated peptides. The two ends of the phosphorylated peptide trapping column are respectively connected with a six-port valve and a ten-port valve. The switching of the ten-port valve can selectively control whether the liquid flowing through the phosphorylated peptide trapping column goes to the waste liquid bottle or the C18 pre-column.

The specific structure is detailed as follows:

The online automated analysis device for phosphoproteomics in this embodiment includes a sample loading part and an analysis part, and the sample loading part includes a sample loading pump 1, a first group of mobile phase storage bottles 15, an automatic sample injection Device 3, Phosphorylated peptide capture column 4, this part is mainly responsible for automatic enrichment of phosphorylated peptides. The analysis part includes the second group of mobile phase storage bottles 16, NC pump (nano-flow pump) 5, C18 pre-column (Thermo Fisher, catalog number AAA-164564) 6, ten-way valve 7, C18 analytical column (Thermo Fisher, Cat. No. 164568)8, nano-ESI ion source 9 and high resolution mass spectrometer 10. The automatic sampler 3 is a device in the prior art, which includes a sample tray 13 , a syringe 14 , a six-way valve 2 and a quantitative loop 11 . The liquid outlet pipe of the first group of mobile phase liquid storage bottles 15 equipped with mobile phase is connected with the liquid inlet pipe of the sample loading pump 1 through the degasser 17. The liquid pipe is connected with the six-way valve 2, the six-way valve 2 is also connected with the inlet end of the phosphorylated peptide capture column 4 through the pipeline, and the six-way valve 2 is provided with a quantitative loop 11, which is connected with the two channels of the six-way valve, The outlet end of the phosphorylated peptide capture column 4 is connected with the ten-way valve 7 through the pipeline, the second group of mobile phase liquid storage bottles 16 are connected with the liquid inlet pipe of the NC pump 5, and the liquid outlet pipe of the NC pump 5 and the ten-way valve 7 pass through. The pipelines are connected, and the ten-way valve 7 is also provided with a C18 pre-column 6, which is connected with the two channels of the ten-way valve 7, and the outlet end of the C18 analysis column can be sequentially connected with the nano ESI ion source 9 and the Orbitrap Fusion mass spectrometer 10, ten The through valve is also connected to a waste liquid bottle 12 through a pipeline. The six-way valve 2 is also connected with a sample tray 13 and a syringe 14 through a pipeline. The sample in the sample tray 13 can enter the quantitative loop of the six-way valve 2 under the push of the syringe 14, and then the valve is cut to make the quantitative loop and the mobile phase pipeline. The mobile phase pushes the sample into the phosphorylated peptide capture column.

The device was used to enrich and analyze the phosphorylated peptides from the mixed peptides obtained after the standard phosphorylated protein α-casein was treated with trypsin, and its sensitivity and reproducibility were investigated. The liquid chromatographic model is DIONEX Ultimate 3000 RSLCnano (Thermo Fisher), which contains a C18 analytical column, and the mass spectrometry model used for the online analysis of phosphorylated peptides is Orbitrap Fusion (Thermo Fisher).

Dissolve 1 mg of standard phosphorylated protein α-casein in 1 ml of 50 mM ammonium bicarbonate, and treat with 20 μg of trypsin for 8 hours to obtain 1 mg/mL of α-casein protease solution. (The standard phosphorylated protein α-casein contains two phosphorylated proteins, α-S1-casein and α-S2-casein, with 9 and 10 phosphorylation sites reported, respectively. After trypsin treatment, a series of Monophosphorylated peptides, polyphosphorylated peptides and non-phosphorylated peptides, the signal of non-phosphorylated peptides was mainly observed in mass spectrometry without specific enrichment). Before use, take 10 μl of 1 mg/mL α-casein protease cleavage solution (80% ACN, 1% TFA) and dilute step by step to obtain 20ng/μl, 10ng/μl, 4ng/μl, 2ng/μl, 0.2ng/μl μl of the α-casein protease digest solution to be analyzed, in the order of the injection volume from low to high.

The specific steps are as follows (Figure 3):

S1. The injection bottles in the autosampler are respectively filled with 0.1M ZrCl ₄ , different concentrations of α-casein protease cleavage solution (dissolved in 80% ACN, 1% TFA), washing solution (80% ACN, 1% TFA), eluent (1M NH ₄ H ₂ PO ₄ ), cleaning solution (40% ACN, 5% NH ₄ OH), edit the chromatographic and mass spectrometry methods of each step in the instrument control software Xcaliur, and order them in the task list Create corresponding analysis tasks. Subsequent S2-S6 are automatically performed under the control of Xcaliur software.

S2. The autosampler sucks 10 microliters of 0.1M ZrCl ₄ into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column under the push of the sampling pump → ten-port valve → waste liquid bottle, the mobile phase of the sampling pump was 0.1% FA, the flow rate was 5 μl/min, and the duration was 6 min. The mobile phase (80% ACN, 0.1% FA) controlled by the NC pump was flowed through the ten-port valve→C18 pre-column→ten-port valve→C18 analytical column for a duration of 6 minutes and the NC pump flow rate was 300 nanoliters/min.

S3. Dissolve the α-casein protease cleavage solution in a solution of 80% ACN and 1% TFA. The autosampler draws 10 microliters of α-casein protease cleavage solution into the quantitative loop, and the six-way valve cuts the valve. Push it to flow through the phosphorylated peptide capture column → ten-port valve → waste liquid bottle, the mobile phase of the sample pump is 0.1% FA, the flow rate is 5 μl/min, and the continuous flushing time is 6 minutes. The mobile phase (80% ACN, 0.1% FA) controlled by the NC pump was flowed through the ten-port valve→C18 pre-column→ten-port valve→C18 analytical column, the NC pump flow rate was 300 nanoliters/min, and the duration was 6 min.

S4. The autosampler draws 10 microliters of washing solution (80% ACN, 1% TFA) into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column driven by the sampling pump → ten-port valve → waste liquid bottle, the loading pump mobile phase was 0.1% FA, the flow rate was 5 μl/min, and the continuous flushing time was 6 minutes. The mobile phase (0.1% FA) controlled by the NC pump was flowed through the ten-port valve→C18 pre-column→ten-port valve→C18 analytical column, and the NC pump flow rate was 300 nanoliters/min for a duration of 6 minutes. Repeat the wash once.

S5. The ten-port valve cuts the valve, the autosampler draws 10 microliters of eluent (1M NH ₄ H ₂ PO ₄ ) into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column driven by the sampling pump → 10-port valve → C18 pre-column → 10-port valve → waste liquid bottle, the mobile phase of the sample pump is 0.1% FA, and the flow rate is 5 μL/min. The mobile phase (0.1% FA) controlled by the NC pump was flowed through a ten-port valve→C18 analytical column with an NC pump flow rate of 300 nanoliters/min. After rinsing for 20 minutes, the ten-port valve was cut, and the mobile phase A of the NC pump (by volume fraction, 0.1% formic acid, 2% acetonitrile) flowed through the ten-port valve → C18 pre-column → ten-port valve → C18 analytical column , continue rinsing and desalting for 10 minutes.

S6. The ten-port valve cuts the valve, the autosampler draws 10 microliters of cleaning solution (40% acetonitrile, 5% NH ₄ OH) into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide captured by the sampling pump Column→10-port valve waste liquid bottle, the mobile phase of the sample pump is 0.1% FA, and the flow rate is maintained at 5 μl/min. At the same time, mass spectrometry detection (Orbitrap Fusion, mass spectrometry parameters are conventional first-level mass spectrometry full scan parameters: spray voltage 2000V, scan range 350-2000m/z, AGC target is 2E5), NC pump-controlled mobile phase (40% ACN, 0.1 %FA) flowed through ten-port valve→C18 pre-column→ten-port valve→C18 analytical column→needle→nano ESI source, the first-order mass spectrum was detected by Orbitrap Fusion mass spectrometer, the NC pump flow rate was 300 nanoliters/min, the upper push the sample pump.

The experimental results are shown in the accompanying drawings 4, 5, 6 and Table 1. The experiment of enriching and analyzing phosphorylated peptides from α-casein shows that the present invention has enrichment effects on mono- and poly-phosphorylated peptides, with a rate as high as 95%. The phosphorylation site coverage (18/19) of the phospho-peptide is as low as 100 fmol, and the detection limit is as low as 100 fmol, and the signal intensity of phosphorylated peptide has a good linear relationship with the loading amount (2ng, 20ng, 40ng, 100ng, 200ng) , and the results obtained from multiple repetitions have very high consistency.

Table 1. Information of phosphorylated peptides identified by the present invention from α-casein digestion

Example 2 On-line analysis of corn whole protease cut

This embodiment makes some improvements on the basis of Embodiment 1, including using actual biological samples, optimizing washing conditions, and using mass spectrometry acquisition parameters suitable for actual samples. The sample used was 300 micrograms of corn whole protease cut. The preparation method of the corn whole protease cut product is as follows: weighing 1 g of corn seedlings developed to the two-leaf stage, grinding to powder with liquid nitrogen; adding 10 ml of extraction buffer (0.1M Tris-Cl (pH=8.0), 10mM EDTA, 0.9M sucrose, 20mM DTT, protease and phosphatase inhibitors (Thermo Fisher, A32961) 2 pieces), after vortex mixing, add 10mL Tris-Cl saturated phenol, after mixing, ice bath sonication (10s on/ 10s stop, 10 cycles); centrifuge (8000g, 4°C, 10min), take the upper phenol phase, add 5 volumes of cold methanol containing 0.1M ammonium acetate, and place at -20°C overnight; centrifuge (8000g, 4°C, 10min), discard the supernatant, wash the protein precipitate with cold methanol, cold acetone, and cold methanol; reconstitute the protein with 320 μL (6M urea, 50mM ammonium bicarbonate); measure the protein concentration by BCA method; add DTT to 10mM, 37°C water bath 1 hour; add iodoacetamide to 40mM, incubate in the dark for 1 hour; the above solution is diluted to 6 times the volume with 50mM ammonium bicarbonate solution, and sequencing grade trypsin is added at a mass ratio of 1/50 enzyme/protein, 37°C Water bath for 12 hours. Use Pierce ^TM polypeptide desalting spin column to remove salt, divide the eluate according to the concentration measured by BCA method according to 1 mg sample/tube, freeze-dry and store at -20°C. Take 1 tube to reconstitute with 66uL (80% acetonitrile, 200 mg/mL 2,5-dihydroxybenzoic acid (DHB), 2% TFA) before use.

Because the amount of injection required to analyze the actual sample is higher and the sample is more complex, this example makes a little improvement in the steps of Figure 3, specifically adding DHB and doubling the concentration of TFA in the sample loading solution and washing solution, This has been documented to enhance selectivity for phosphorylated peptides, followed by removal of DHB residues with DHB-free Wash 2.

Specific steps are as follows:

S1. The injection bottles in the autosampler were respectively filled with 0.1M ZrCl ₄ , corn seedling whole protein enzyme digestion solution (1 mg dissolved in 60 μL (80% acetonitrile, 200 mg/mL DHB, 2% TFA), washing solution 1 (80% acetonitrile, 200 mg/mL DHB, 2% TFA), washing solution ₂ (80% acetonitrile, 1% TFA), eluent (1M _NH4H2PO4 ), washing solution (40% ACN, 5% NH) ₄ OH), edit the chromatographic and mass spectrometry methods of each step in the instrument control software Xcaliur, and set up the corresponding analysis tasks in the task list in turn. The subsequent S2-S6 are automatically carried out under the control of the Xcaliur software.

S2. The autosampler sucks 10 microliters of 0.1M ZrCl4 into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column under the push of the sampling pump → ten-port valve → waste liquid bottle. The mobile phase of the sampling pump is 0.1% FA at a flow rate of 5 μl/min for a duration of 6 min. The mobile phase (80% ACN, 0.1% FA) controlled by the NC pump was flowed through the ten-port valve→C18 pre-column→ten-port valve→C18 analytical column for a duration of 6 minutes and the NC pump flow rate was 300 nanoliters/min.

S3. The autosampler sucks 20μL of corn seedling whole protein enzyme digestion solution (1mg is dissolved in 60μL (80% acetonitrile, 200mg/mL DHB, 2% TFA) into the quantitative loop, the six-way valve cuts the valve, and the sample pump pushes down the flow The phosphorylated peptide capture column → ten-port valve → waste liquid bottle, the mobile phase of the loading pump is 0.1% FA, the flow rate is 5 μl/min, and the continuous flushing time is 6 minutes. The mobile phase controlled by the NC pump (80% ACN , 0.1% FA) through the ten-way valve→C18 pre-column→ten-way valve→C18 analytical column, the NC pump flow rate is 300 nanoliters/min, and the duration is 6 minutes.

S4. The autosampler draws 10 microliters of washing solution 1 (80% acetonitrile, 200 mg/mL DHB, 2% TFA) into the quantitative loop, the six-way valve cuts the valve, and flows through the phosphorylated peptide capture column driven by the sampling pump → Ten-way valve → waste liquid bottle, the mobile phase of the sample pump is 0.1% FA, the flow rate is 5 μl/min, and the continuous flushing time is 6 minutes. The mobile phase (0.1% FA) controlled by the NC pump was flowed through the ten-port valve→C18 pre-column→ten-port valve→C18 analytical column, and the NC pump flow rate was 300 nanoliters/min for a duration of 6 minutes. Washing solution 1 was repeated once, and washing solution 2 (80% acetonitrile, 1% TFA) was repeated once.

S5. The ten-port valve cuts the valve, the autosampler draws 10 microliters of eluent (1M NH ₄ H ₂ PO ₄ ) into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column driven by the sampling pump → 10-port valve → C18 pre-column → 10-port valve → waste liquid bottle, the mobile phase of the sample pump is 0.1% FA, and the flow rate is 5 μL/min. The mobile phase (0.1% FA by volume fraction) controlled by an NC pump was passed through a ten-way valve→C18 analytical column at a flow rate of 300 nanoliters/min. After rinsing for 20 minutes, the ten-port valve was cut, and the mobile phase A of the NC pump (by volume fraction, 0.1% formic acid, 2% acetonitrile) flowed through the ten-port valve → C18 pre-column → ten-port valve → C18 analytical column , continue rinsing and desalting for 10 minutes.

S6. The ten-port valve cuts the valve, the autosampler sucks 10 microliters of cleaning solution (40% acetonitrile, 5% NH4OH) into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column driven by the sampling pump → Ten-way valve waste bottle, the mobile phase of the sample pump is 0.1% FA, and the flow rate is maintained at 5 μl/min. At the same time, mass spectrometry detection (Orbitrap Fusion, mass spectrometry parameters are conventional phosphorylated proteomics analysis parameters, spray voltage 2000V, ion transfer tube temperature 320°C, MS detector is Orbitrap, resolution 120k, MSMS detector is Ion Trap, Scan rate is Rapid), 120-minute acetonitrile gradient mobile phase (4-32% ACN, 0.1FA) controlled by NC pump flows through ten-port valve→C18 pre-column→ten-port valve→C18 analytical column→needle→nano ESI source , with a flow rate of 300 nanoliters/min.

S7. Use Proteome Discoverer 2.4 software to identify the RAW file of the mass spectrometer, select the Zea mays (UP000007305) proteome FASTA file downloaded from Uniprot as the database, set (S, T, Y) phosphorylation to variable modification, before The mass tolerance of bulk ions was 10 ppm, and the mass tolerance of fragment ions was 0.2 Da. Percolator was used for quality control, and the FDR value was set to 0.01.

The experimental results show that a total of 1747 phosphorylated peptides, 2129 phosphorylation sites, and 1173 phosphorylated proteins were identified in this analysis, which is very ideal.

Claims (10)

1. An on-line automated analysis device for phosphorylation proteomics, which is a liquid chromatography-mass spectrometer, the liquid chromatography includes a phosphorylated peptide capture column and an analysis column, wherein the phosphorylated The peptide capture column is an ATP-modified immobilized metal ion affinity chromatography column;

   The preparation method of the ATP-modified immobilized metal ion affinity chromatographic column is:

   Mix and stir potassium water glass, γ-glycidyl etheroxypropyltrimethoxysilane and water-dissolved adenosine triphosphate disodium, then add water-dissolved formamide and stir to obtain a reaction solution, fill the column with the reaction solution, and then fill The reaction solution of the chromatographic column is reacted and solidified, and then washed to obtain an ATP-modified immobilized metal ion affinity chromatographic column.
2. The on-line automatic analysis device according to claim 1, wherein the dosage-mass ratio of the potassium water glass, γ-glycidyl ether oxypropyltrimethoxysilane, adenosine triphosphate disodium salt and formamide is 500- 2000:1-10:2-50:20-130.
3. On-line automated analysis device according to claim 2, is characterized in that, the consumption mass ratio of described potassium water glass, γ-glycidyl ether oxypropyl trimethoxysilane, adenosine triphosphate disodium salt and formamide is 1000: 6:7.5:68; the modulus range of the potassium water glass is 2-4, and the Baumé degree range is 20-50.
4. The online automatic analysis device according to claim 3, wherein the modulus of the potassium water glass is 3.3, and the Baumé degree is 40.
5. The online automatic analysis device according to claim 1, wherein the curing is curing at a temperature of 100° C. for 10 hours; and the washing is successively washed with 1M ammonium nitrate, 0.1M nitric acid and water.
6. The online automated analysis device according to claim 1, wherein the chromatographic column is an elastic quartz capillary.
7. On-line automatic analysis device according to claim 6, is characterized in that, described elastic quartz capillary is the elastic quartz capillary of outer diameter 360 microns, inner diameter 150 microns, length 15 centimeters
8. The online automatic analysis device according to claim 1, 2, 3, 4, 5, 6 or 7, characterized in that, the online automatic analysis device for phosphorylation proteomics comprises a sample loading part and an analysis The sample loading part includes a first group of mobile phase storage bottles, a second group of mobile phase storage bottles, a degasser, a sample loading pump, an automatic sampler, and a phosphorylated peptide capture column. The analysis Parts include NC pump, C18 pre-column, ten-port valve, C18 analytical column, nano-ESI and high-resolution mass spectrometer, the first set of mobile phase storage bottles, degasser, sample pump, autosampler The six-port valve, the phosphorylated peptide capture column and the ten-port valve are connected in sequence through pipelines. The six-port valve is also provided with a quantitative loop connected to the two channels of the six-port valve, and the autosampler is also provided with a sample tray and The syringes are respectively connected with the six-way valve, the second group of mobile phase storage bottles, the NC pump and the ten-way valve are connected in sequence through the pipeline, and a C18 pre-column is also provided on the ten-way valve, and the C18 pre-column is connected with the ten-way valve through the pipeline. , the ten-way valve is also connected with the C18 analytical column, the outlet end of the C18 analytical column can be sequentially connected with the nano-ESI and high-resolution mass spectrometer, and the ten-way valve is also connected with a waste liquid bottle.
9. An analytical method for phosphorylation proteomics, characterized in that it comprises the following steps:

   A. The phosphorylated peptide capture column is eluted with ZrCl ₄ , so that the ATP-modified immobilized metal ion affinity chromatography column packing chelates Zr ⁴⁺ ;

   B, take the sample to flow through the phosphorylated peptide capture column of step A to complete the enrichment of phosphorylated peptides;

   C, washing liquid flows through the phosphorylated peptide capture column of step B, completes the cleaning to non-specific binding polypeptide;

   D. The eluate flows through the phosphorylated peptide capture column of step B, and the enriched phosphorylated peptides are eluted, and the eluted phosphorylated peptides enter the C18 pre-column;

   E. The C18 pre-column is eluted with the mobile phase, and the phosphorylated peptide enters the C18 analytical column and is identified by the mass spectrometer.
10. Analysis method according to claim 9, is characterized in that, using the online automation analysis device described in claim 8, the step is:

    S1. 0.1M ZrCl ₄ is respectively placed in the injection bottle in the autosampler; sample solution; washing solution: 80% ACN, 1% TFA by volume fraction; eluent: 1M NH ₄ H ₂ PO ₄ ; washing solution : volume fraction 40% ACN, 5% NH ₄ OH;

    S2. The automatic sampler sucks 0.1M ZrCl ₄ into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column under the push of the sample pump → ten-port valve → waste liquid bottle, the mobile phase of the sample pump is the volume fraction 0.1% FA, the mobile phase volume fraction controlled by NC pump is 80% ACN, 0.1% FA flows through the ten-way valve→C18 pre-column→ten-way valve→C18 analytical column;

    S3. Dissolve the sample in a solution with a volume fraction of 80% ACN and 1% TFA, the automatic sampler draws the sample solution into the quantitative loop, the six-way valve cuts the valve, and flows through the phosphorylated peptide capture column driven by the sampling pump → ten Pass valve → waste liquid bottle, the mobile phase of the loading pump is 0.1% FA, and the mobile phase controlled by the NC pump is 80% ACN and 0.1% FA flow through the ten-way valve → C18 pre-column → ten-way valve → C18 analysis column;

    S4. The automatic sampler draws the washing liquid fraction of 80% ACN and 1% TFA into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column under the push of the sample pump → ten-port valve → waste liquid bottle, upper The mobile phase of the sample pump is 0.1% FA in volume, and the mobile phase controlled by the NC pump with a volume fraction of 0.1% FA flows through the ten-port valve → C18 pre-column → ten-port valve → C18 analytical column;

    S5. The ten-port valve cuts the valve, the autosampler draws the eluent 1M NH ₄ H ₂ PO ₄ into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column driven by the sample pump → ten-port valve → C18 pre-column → ten-port valve → waste liquid bottle, the mobile phase of the loading pump is 0.1% FA by volume; the mobile phase controlled by the NC pump with a volume fraction of 0.1% FA flows through the ten-port valve → C18 analytical column; after elution, ten Open the valve and cut the valve, let the mobile phase A of the NC pump with a volume fraction of 0.1% formic acid and 2% acetonitrile flow through the ten-port valve → C18 pre-column → ten-port valve → C18 analytical column, and continue to rinse and remove salt;

    S6. The ten-port valve cuts the valve, the automatic sampler absorbs the cleaning liquid fraction of 40% acetonitrile and 5% NH ₄ OH into the quantitative loop, the six-port valve cuts the valve, and flows through the phosphorylated peptide capture column driven by the sampling pump → ten Open valve waste liquid bottle, the mobile phase of the loading pump is 0.1% FA, and the mass spectrometry detection is turned on; the mobile phase volume controlled by the NC pump is 40% ACN, 0.1% FA flows through the ten-way valve → C18 pre-column → ten-way Valve→C18 analytical column→needle→nano ESI source, detected with mass spectrometer.

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