CN111521473A

CN111521473A - Device for preparing proteomics micro samples

Info

Publication number: CN111521473A
Application number: CN202010351887.4A
Authority: CN
Inventors: 应万涛; 钱小红; 孟波; 李晓宇
Original assignee: BEIJING PROTEOME RESEARCH CENTER; Institute of Pharmacology and Toxicology of AMMS
Current assignee: BEIJING PROTEOME RESEARCH CENTER; Institute of Pharmacology and Toxicology of AMMS; Academy of Military Medical Sciences AMMS of PLA
Priority date: 2020-04-28
Filing date: 2020-04-28
Publication date: 2020-08-11

Abstract

The invention discloses a device for preparing proteomics micro samples. The device is simple to manufacture and low in cost, and the recovery rate of the peptide fragment and the protein identification amount are superior to those of the conventional method under the condition of low initial protein amount. The device realizes seamless combination of one-stop in-situ protein digestion, peptide fragment acquisition, N-sugar chain release and enrichment. The loss of the sample is reduced to the maximum extent, and the complete process sample preparation of the micro sample proteomics is realized.

Description

Device for preparing proteomics micro samples

Technical Field

The invention belongs to the field of proteomics, and relates to a device for preparing proteomic micro samples.

Background

The reversed phase chromatography is an elution chromatography method which uses a nonpolar reversed phase medium as a stationary phase and an aqueous solution of a polar organic solvent as a mobile phase to separate and purify solutes according to the difference of the polarity (hydrophobicity) of the solutes. In reverse phase chromatography, solutes are distributed to the stationary phase surface by hydrophobic interactions, but strong hydrophobicity is exhibited since the stationary phase surface is completely covered with non-polar groups. Therefore, it is necessary to perform elution separation of the solute with a polar organic solvent (e.g., methanol, acetonitrile, etc.) or an aqueous solution thereof.

Disclosure of Invention

The invention aims to provide a device for proteomics micro sample preparation. It is named (Microsample preparation technology, abbreviated as Micro-SPAT). The device has simple and quick manufacturing method and low cost, and can be operated in a conventional laboratory. Can be used for research of proteomics and glycomics of submicron protein. The device realizes in-situ enzyme digestion of protein and satisfies seamless combination of elution and enrichment of N-sugar.

The device for preparing the micro-sample comprises C18-Tip and HILIC-Tip;

the C18-Tip is capable of achieving protein digestion and N-glycan release;

the HILIC-Tip is capable of enriching N-glycans.

In the above apparatus, said C₁₈Tip comprises two layers of C8 film with C18 filler between them; two layers of C8 membranes can isolate the reaction chamber;

the HILIC-Tip comprises a C8 film and a HILIC filler on the C8 film;

specifically, the C18-Tip and the HILIC-Tip both also comprise a container for placing the components; the container is in particular a pipette tip (Axygen);

the mass of the C18 filler, in terms of protein: c18 ═ 10 μ g: 1 mg;

the HILIC filler is prepared from the following components in percentage by weight: HILIC ═ 10 μ g: 0.5 mg.

The HILIC-Tip can purify the N-glycans by using the hydrophilic and hydrophobic interactions of HILIC fillers.

Specifically, the C18-Tip is activated as follows:

to the C18-Tip were added ACN, 50% ACN + 0.1% aqueous TFA (v/v) and 0.1% aqueous TFA (v/v) in this order, and centrifuged to remove the liquid.

The dosage of the activating agent used for each activation is 100 mul;

in the centrifugation step, the centrifugal force is 500-2000 g; in particular 1500 g;

centrifuging with the ACN for 20-60 s; specifically 30 s;

the time of centrifugation with the (50% ACN + 0.1% TFA) aqueous solution is 50-100 s; specifically 60 s;

the time for centrifugation with the 0.1% aqueous TFA solution is 80-150 s; specifically 90 s;

the ACN represents pure acetonitrile;

the TFA represents trifluoroacetic acid.

The HILIC-Tip was activated as follows:

adding 100 μ l of 1% TFA solution to the HILIC-Tip, centrifuging at 1500g for 90s, removing liquid, and repeating for 3 times;

then, 100. mu.l of 80% ACN + 1% TFA solution was added to the HILIC-Tip, and the mixture was centrifuged at 1500g for 60 seconds to remove the liquid, which was repeated 3 times.

In addition, the application of the device provided by the invention in the preparation of micro samples also belongs to the protection scope of the invention.

In the above application, specifically, the sample is a cell, a tissue or a body fluid.

The invention provides a method for preparing a trace sample proteomics full-process sample, which comprises the following steps:

1) digesting the protein into peptide fragments in the C18-Tip of the device, releasing N-sugar chains, centrifuging, desalting the peptide fragments or eluting the N-sugar chains, and then adding a flushing mobile phase which is not the N-sugar chains for centrifuging and eluting the peptide fragments;

2) placing the N-sugar chain-containing substance treated in the step 1) in the HILIC-Tip, centrifuging to remove non-N-sugar chains, and enriching N-sugar chains.

Said step 1) of the above method washes the non-N-sugar chain mobile phase consisting of water, acetonitrile and trifluoroacetic acid; the volume ratio of the water to the acetonitrile to the trifluoroacetic acid is 20:80:0.1-1 or 20:80: 0.5; specifically 20:80: 1;

the desalting/eluting mobile phase of the N-sugar chain is an aqueous solution of trifluoroacetic acid; specifically, the aqueous solution of trifluoroacetic acid with the volume percentage concentration of 0.1 percent;

the mobile phase for eluting the peptide fragment consists of water, acetonitrile and trifluoroacetic acid; the volume ratio of the water to the acetonitrile to the trifluoroacetic acid is (50-20): (50-80): 0.1; specifically, 50: 50: 0.1.

in the step 1) and the step 2), the centrifugal force is 500-2000 g; in particular 1500 g; the time is 20-90 s; specifically 30 s.

The invention provides a small column filled with reversed phase chromatographic packing (C18) as a reaction chamber for protein digestion, and the characteristic of C18 packing is adopted to realize the cleaning of peptide fragments; the small column is filled with hydrophilic interaction chromatographic packing (HILIC), and the hydrophilicity of the HILIC packing is adopted to enrich the N-glycan, so that the sample preparation requirement of protein N-glycome analysis of the submicron protein is met.

The invention has the beneficial effects that:

1) the device provided by the invention greatly reduces the initial amount of protein for sample preparation, and meets the proteomics research requirements of samples with less protein amount clinically.

2) The recovery rate of peptide fragments and the identification amount of protein are better than those of the conventional method under the condition of less protein.

3) The peptide fragment cleavage rate is lower than 20%, and the method can be effectively used for quantitative analysis of data.

4) The method carries out protein research deeply to the N-carbohydrate group level, and realizes the sample preparation of the sub-microgram protein N-carbohydrate group analysis.

The device provided by the invention 5) is simple to manufacture, low in cost and easy to operate, and can be used for research of proteomics and glycomics of sub-microgram protein. The device realizes in-situ enzyme digestion of protein and satisfies seamless combination of elution and enrichment of N-sugar. Has excellent performance in the aspect of proteomics sample preparation and has important application value.

Drawings

Figure 1 is a simplified diagram of a proteomics micro sample preparation device.

FIG. 2 is a histogram of the results of 3 replicates of the whole protein proteome analysis of 10. mu.g HepG2 cell line protein and peptide fragment identification.

FIG. 3 is a Pearson correlation analysis of 10 μ g HepG2 cell line whole protein proteome analysis for 3 replicates.

FIG. 4 is a histogram of peptide truncation ratio of 3 replicates of the 10 μ g HepG2 cell line whole protein proteome analysis.

FIG. 5 is a MALDI-TOF MS spectrum of N-sugar chains obtained from 10. mu.g IgG using Micro-SPAT.

FIG. 6 shows MALDI-TOF MS spectra of 10. mu.g of HepG2 cell-derived protein obtained by preparing N-sugar chains by Micro-SPAT method.

FIG. 7 is a correlation analysis of 3 replicates of the holoprotein N-glycome analysis of the 10. mu.g HepG2 cell line.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified. The reversed phase chromatographic packing C18 used in the invention was purchased from Tianjin Bonner Aijiel scientific Co; the hydrophilic interaction chromatography packing HILIC is purchased from Tianjin Borna Aijiel scientific company; the C8 film used was purchased from 3M company.

Example 1 fabrication and activation of proteomics micro sample preparation device

(1) C18-Tip preparation: a layer of C8 film was added to the 200. mu.l pipette tip as a sieve plate, 1mg of C18 filler was introduced above the sieve plate, and finally a layer of sieve plate was added above the C18 filler.

(2) C18-Tip activation as described above: first, 100. mu.l of pure acetonitrile (ACN, Merck) was added to C18-Tip, and centrifuged at 1500g for 30s to remove the liquid; next, 100. mu.l of a 50% ACN + 0.1% TFA (trifluoroacetic acid, Sigma) solution was added to Tip, and centrifuged at 1500g for 60s to remove the liquid; finally, 100. mu.l of 0.1% TFA solution was added to C18-Tip, and centrifuged at 1500g for 90s to remove the liquid.

(3) Preparing HILIC-Tip: a layer of C8 film was added to 200. mu.l of pipette tip as sieve plate, and HILIC filler 0.5mg was introduced above the sieve plate to make enrichment column.

(4) Activation of HILIC-Tip as described above: first, 100. mu.l of 1% TFA solution was added to HILIC-Tip, and centrifuged at 1500g for 90s to remove the liquid, which was repeated 3 times; then, 100. mu.l of 80% ACN + 1% TFA solution was added to HILIC-Tip, and the mixture was centrifuged at 1500g for 60 seconds to remove the liquid, which was repeated 3 times.

The proteomics micro sample preparation device provided by the present invention was fabricated according to the experimental conditions of example 1.

FIG. 1 is a simplified diagram of the proteomic micro sample preparation device of this example. The device is composed of C18-Tip and HILIC-Tip, and the C18 filler introduced in the C18-Tip is reverse filler which can be used for washing peptide fragments or separating the peptide fragments from N-glycan. Hydrophilic filler is introduced into HILIC-Tip, so that the effect of enriching N-glycan is achieved. The device takes C18-Tip as a reaction chamber for protein digestion and N-glycan release, so that the loss of a sample is reduced to the maximum extent, and HILIC enrichment can be directly performed after N-glycan elution. Thereby achieving seamless combination of N-glycan elution and enrichment.

Example 2 human hepatoma HepG2 cell line Total protein digestion experiment

The human hepatoma HepG2 cell line (purchased from American model culture Collection library) whole protein was extracted with 8M urea (UA, Sigma) solution and the protein concentration was measured by the Bradford method.

Mu.g of protein was taken and placed in C18-Tip, tris (2-carboxyethyl) phosphine (TCEP, Sigma) was added to a final concentration of 10mM, chloroacetamide (CAA, Sigma) was added to a final concentration of 15mM, the mixture was flushed well and subjected to reduction and alkylation reactions simultaneously at 37 ℃ for 1 h. Both TCEP and CAA were solubilized using 8M UA.

Intracellular proteases (Lys-C, Spec. Act:14.6 Unit/(min. mg), 250 mM. 2775. mu.l Ac-Lys-pNA and 20. mu.g/mL. 225. mu.l protease) were added to the solution at an enzyme/protein ratio of 1:100(w/w), incubated at 30 ℃ for 30min and the absorbance at 405nm (mOD/min) was measured over time, the maximum slope of the curve was used for activity calculation, the Unit being defined as 1Unit to 1OD₄₀₅And/min. ) And then 50mM hydrogen carbonate (NH) is supplemented₄HCO₃) Ammonium to make the UA concentration in the system 6M, and incubating for 6h at 37 ℃ to digest HepG2 cell line holoprotein enzymatically.

50mM NH was added₄HCO₃The HepG2 cell line holoprotein was digested enzymatically by adding Trypsin (Trypsin, Spec.Act:16987u/mg, enzyme activity unit per mg of protein) to the solution at an enzyme/protein ratio of 1:10(w/w) to a UA concentration of less than 1M and incubating at 37 ℃ for 16 h.

The resulting solution was centrifuged at 1500g for 3min with TFA added to adjust the concentration of TFA in the solution to 0.1%, and the solution was removed.

Add the same volume of 0.1% TFA solution as the cleavage solution, centrifuge at 1500g for 3min, repeat 3 times.

Adding 100 μ l 50% ACN + 0.1% TFA solution to elute peptide fragment, repeating for three times, collecting eluate, and heat drying in a vacuum heat drying machine at 45 deg.C, and storing at-20 deg.C for use.

Redissolving the peptide fragment with 0.1% FA solution, measuring the concentration of the peptide fragment by using Nanodrop, and analyzing 500ng by using a liquid chromatography mass spectrometer.

10 ug of HepG2 cell line holoprotein was digested according to the experimental conditions of example 2 and subjected to mass spectrometry using LC-MS, as shown in FIG. 2, which is a histogram of the results of three replicates of the 10 ug HepG2 cell line holoprotein proteome analysis of the present example, identifying the protein and peptide fragments. It can be seen from the figure that the peptide fragment identification amount is about 11000 and the protein identification amount is about 2200 in three repeated experiments. This sample was analyzed using a QE-PLUS mass spectrometer and the library was searched by Maxquant software for the mass spectrometer output results.

FIG. 3 shows the Pearson correlation analysis of triplicates of the 10. mu.g HepG2 cell line holoprotein proteome analysis of this example. From the figure, it can be seen that the correlation analysis R of the Pearson in the three repeated experiments²>0.98, which shows that the correlation among three repeated experiments is very good.

FIG. 4 shows the peptide truncation histogram of the 10. mu.g HepG2 cell line holoprotein proteome analysis of the example in three replicates. The result shows that the peptide fragment missing cut in three repeated experiments is about 15%, and the result can completely meet the requirement of quantitative analysis of the peptide fragment.

The above results indicate that the device has utility in proteome analysis of micro-complex biological samples.

Example 3 human immunoglobulin G (IgG) sugar cleavage and enrichment experiments

1) Human immunoglobulin G (IgG) dissolved in 50mM NH₄HCO₃In (1).

2) Mu.g of IgG was taken in C18-Tip, tris (2-carboxyethyl) phosphine (TCEP) was added to a final concentration of 10mM, Chloroacetamide (CAA) was added to a final concentration of 15mM, and the mixture was mixed by pipetting, and reduction and alkylation were simultaneously carried out at 37 ℃ for 1 hour.

3) Trypsin was added to the solution at an enzyme/protein ratio of 1:100(w/w) and incubated at 37 ℃ for 16h to enzymatically digest IgG.

4) peptide-N-glycosidase F (PNGase F, Activity: >1000u/mg, enzyme Activity units per mg of protein) was added to the solution in an enzyme/protein ratio of 1:20 (w/w). ) The N-sugar chains were released by incubating at 37 ℃ for 2 hours.

5) After adding TFA to adjust the concentration of TFA in the cleavage solution to 0.1%, 1500g of the resulting solution was centrifuged for 3min to collect the N-sugar chain.

6) Adding 0.1% TFA solution with the same volume as the enzyme solution, centrifuging at 1500g for 3min, repeating for 3 times, and collecting N-sugar chain.

7) The N-sugar chains collected in the two steps (5) (6) were combined and added with neat ACN and TFA to give 80% ACN + 1% TFA.

8) The final liquid in (7) was added to HILIC-Tip, centrifuged at 1500g for 30s, enriched with N-sugar chains, and repeated 3 times.

9) HILIC-Tip was washed once with 100. mu.l of 80% ACN + 1% TFA solution.

10) The N-sugar chain was eluted with 100. mu.l of 0.1% TFA solution and repeated 3 times.

11) TFA was added to give a TFA concentration of 0.22%, and the sialic acid was removed by cooking at 82 ℃ for 2 h.

12) Vacuum heat drying at 45 ℃, redissolving N-sugar chains with 2 μ l of water, and analyzing the N-sugar chain sample by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS).

N-sugar chains of 10. mu.g of IgG were prepared according to the experimental conditions of example 3 above and subjected to mass spectrometry using MALDI-TOF-MS, as shown in FIG. 5, which is a MALDI-TOFMS spectrum of N-sugar chains of 10. mu.g of IgG obtained using Micro-SPAT. As can be seen from the figure, 20N-sugar chains were obtained from 10. mu.g of IgG by this technique based on MALDI-TOF-MS.

The above results indicate that the technique has utility in the analysis of N-glycome of minute quantities of simple biological samples.

Example 4 human hepatoma HepG2 cell line Total protein sugar cutting and enrichment experiments

1) The human liver cancer HepG2 cell line holoprotein was extracted using 8M UA and the protein concentration was measured using the Bradford method.

2) 10 μ g of protein was taken in C18-Tip, tris (2-carboxyethyl) phosphine (TCEP) was added to a final concentration of 10mM, Chloroacetamide (CAA) was added to a final concentration of 15mM, and the mixture was stirred and mixed well, and reduction and alkylation reactions were simultaneously carried out at 37 ℃ for 1 hour.

3) Trypsin was added to the solution at an enzyme/protein ratio of 1:100(w/w) and incubated at 37 ℃ for 16h to enzymatically digest the human liver cancer HepG2 cell line holoprotein.

4) PNGase F was added to the solution at an enzyme/protein ratio of 1:20(w/w), and incubated at 37 ℃ for 2h to release the N-sugar chains.

9) HILIC-Tip was washed once with 100. mu.l of 80% ACN + 1% TFA solution.

12) Vacuum heat-dried at 45 ℃ and 2. mu.l of water redissolved the N-sugar chains, and the N-sugar chain samples were analyzed by MALDI-TOF MS.

N-sugar chains were prepared from 10. mu.g of HepG2 according to the experimental conditions of example 4 and subjected to mass spectrometry using MALDI-TOFMS, as shown in FIG. 6, which is a MALDI-TOFMS spectrum of N-sugar chains obtained from 10. mu.g of HepG2 using Micro-SPAT. As can be seen from the figure, 39N-sugar chains were obtained from 10. mu.g of HepG2 by this technique based on MALDI-TOF-MS. And the same number of glycoforms of the same type were obtained in all 3 replicates.

FIG. 7 shows the correlation analysis of the results of 3 replicates of the N-glycome analysis of 10. mu.g HepG2 cell extract protein. As can be seen from the figure, the correlation between Pearson and R was good in 3 replicates²＝0.953-0.976。

The above results indicate that the device for proteomic micro sample preparation provided by the present invention has utility in N-glycome analysis of micro complex biological samples.

Claims

1. A device for micro-scale sample preparation comprises C18-Tip and HILIC-Tip;

the C18-Tip is capable of achieving protein digestion and N-glycan release;

the HILIC-Tip is capable of enriching N-glycans.

2. The apparatus of claim 1, wherein: said C is₁₈Tip comprises two layers of C8 film with C18 filler between them;

the HILIC-Tip comprises a C8 film and a HILIC filler on the C8 film;

specifically, the C18-Tip and the HILIC-Tip both also comprise a container for placing the components; the container is specifically a pipette tip;

the mass of the C18 filler, in terms of protein: c18 ═ 10 μ g: 1 mg;

3. The apparatus of claim 1 or 2, wherein: the C18-Tip was activated as follows:

4. The apparatus of claim 3, wherein: the dosage of the activating agent used for each activation is 100 mul;

centrifuging with the ACN for 20-60 s; specifically 30 s;

the ACN represents pure acetonitrile;

the TFA represents trifluoroacetic acid.

5. The apparatus of any of claims 1-4, wherein: the HILIC-Tip was activated as follows:

6. Use of the device according to any one of claims 1 to 5 for micro-sample preparation.

7. Use according to claim 6, characterized in that: the sample is a cell, tissue or body fluid.

8. A method for whole-flow sample preparation of micro-sample proteomics, comprising:

1) in the C18-Tip according to any one of claims 1 to 5, digesting the protein into a peptide fragment, releasing N-sugar chains, centrifuging, desalting the peptide fragment or eluting the N-sugar chains, and further centrifuging the eluted peptide fragment with a mobile phase which washes non-N-sugar chains;

9. The method of claim 8, wherein: the step 1) washing the non-N-sugar chain mobile phase consists of water, acetonitrile and trifluoroacetic acid; the volume ratio of the water to the acetonitrile to the trifluoroacetic acid is 20:80:0.1-1 or 20:80: 0.5; specifically 20:80: 1;

10. the method according to claim 8 or 9, characterized in that: in the step 1) and the step 2), the centrifugal force is 500-2000 g; in particular 1500 g; the time is 20-90 s; specifically 30 s.