CN111721939B - Pretreatment method and treatment kit for mass spectrometry detection of protein gel sample - Google Patents
Pretreatment method and treatment kit for mass spectrometry detection of protein gel sample Download PDFInfo
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Abstract
The invention discloses a mass spectrometry pretreatment method and a treatment kit for a protein gel sample. The method comprises (1) enzymolysis in rubber: after the lane part to be detected of protein gel electrophoresis is finely crushed, washing, protein reduction treatment, protein alkylation treatment and protease enzymolysis treatment are carried out, and then acidic acetonitrile solution is adopted to carry out peptide segment extraction, so as to obtain peptide segment-acidic acetonitrile solution; (2) peptide fragment desalting: transferring the peptide-acetonitrile solution to a separation column filled with reversed phase chromatographic packing with cation exchange function, desalting and washing with trifluoroacetic acid with mass fraction of 0.1% -0.5%, eluting with alkaline acetonitrile solution, and collecting eluent containing peptide as sample for mass spectrometry detection. The method comprises the following steps: peptide extraction liquid, desalting cleaning liquid and desalting eluent. The invention reduces the requirement on experimental conditions and shortens the sample preparation time to about half.
Description
Technical Field
The invention belongs to the technical field of proteomics detection, and particularly relates to a mass spectrometry detection pretreatment method and a treatment kit for a protein gel sample.
Background
At present, protein samples for mass spectrometry are generally prepared by carrying out in-gel enzymolysis and desalination treatment on protein gel samples after gel electrophoresis separation, and then carrying out mass spectrometry detection. The pretreatment of mass spectrum detection comprises the following steps: firstly, cutting up gel blocks, sequentially carrying out reduction and alkylation treatment on proteins by Dithiothreitol (DTT) and Iodoacetamide (IAA), and then extracting peptide solution after pancreatin enzymolysis; after that, the peptide fragment solution is pumped down by a vacuum centrifugal concentrator, and then the peptide fragment is dissolved by an acid solution to realize solvent replacement, and then the solution is transferred to a C18 separation column for desalination, and finally the peptide fragment is eluted by a high organic phase for mass spectrum detection.
The prior pretreatment process of protein mass spectrometry detection has a plurality of steps, and particularly needs to use instruments which are not configured in conventional laboratories such as a vacuum centrifugal concentrator, the whole sample preparation process consumes between 20 and 30 hours, and meanwhile, due to the configuration problem of the instruments, protein or peptide fragment samples possibly need to be transported to the laboratory with corresponding conditions, so that serious preservation and transportation problems are brought. Thus, is not conducive to large-scale, commercial detection.
Disclosure of Invention
Aiming at the defects or improvement demands of the prior art, the invention provides a pretreatment method and a treatment kit for mass spectrometry detection of protein gel samples, which aim at realizing a peptide segment desalting step by adopting reverse phase chromatographic packing with cation exchange effect without solvent replacement, avoiding a solvent pumping step by adopting a vacuum centrifugal concentrator, reducing the requirements on experimental conditions, and greatly shortening the preparation time of the protein gel mass spectrometry detection samples, thereby solving the technical problems of higher requirements on the experimental conditions and long treatment time of the protein mass spectrometry detection samples in the prior art.
In order to achieve the above object, according to one aspect of the present invention, there is provided a method for mass spectrometry pretreatment of a protein gel sample, comprising the steps of:
(1) Enzymolysis in the glue: after the lane part to be detected of protein gel electrophoresis is finely crushed, washing, protein reduction treatment, protein alkylation treatment and protease enzymolysis treatment are carried out, and then acidic acetonitrile solution is adopted to carry out peptide segment extraction, so as to obtain peptide segment-acidic acetonitrile solution; the acidic acetonitrile solution contains 3 to 10 percent of formic acid, and the acetonitrile concentration is between 50 and 80 percent;
(2) Desalting the peptide: transferring the peptide-acetonitrile solution obtained in the step (1) into a separation column filled with reversed phase chromatographic packing with cation exchange function, desalting and washing by using trifluoroacetic acid solution with mass fraction of 0.1% -0.5%, eluting by using alkaline acetonitrile solution, and collecting eluent containing the peptide as a mass spectrum detection sample.
Preferably, the reverse phase chromatographic packing has cation exchange function, when the pH value of the solution is acidic, the peptide segment has positive charges so as to be firmly adsorbed with the reverse phase chromatographic packing, after desalination and washing, the pH value of the environment is changed through eluent so that the peptide segment does not have positive charges any more, and is eluted in alkaline eluent, and the preferred reverse phase chromatographic packing is SDB-RPS reverse phase chromatographic packing, which is a styrene-divinylbenzene copolymer modified by sulfonic groups.
Preferably, the protein gel sample is subjected to pretreatment for mass spectrometry, wherein the basic acetonitrile solution contains 1% -3% of NH 4 OH, acetonitrile concentration between 60% and 90%.
Preferably, the pretreatment method for mass spectrometry detection of the protein gel sample comprises the following specific steps of: adding 120-180 mu L of solution containing 0.1% -0.5% trifluoroacetic acid into a separation column for adsorbing peptide fragments to be desalted, and passing through the column to obtain a liquid; preferably, the washing operation is repeated 1 to 3 times;
the operation of eluting with basic acetonitrile solution is specifically as follows: adding 15-20 mu L of basic acetonitrile solution to a separation column adsorbed with desalted peptide fragments, and collecting a liquid phase after passing through the column; preferably the elution operation is repeated 2 to 3 times.
Preferably, the separation column is comprised in a peptide fragment desalting device. The peptide fragment desalting device consists of a detachable nested separation column, an adapter and a collecting pipe. The separation column is a conical tube with two open ends, and the position 3 to 8mm away from the lower end is filled with reversed phase chromatographic packing; the collecting pipe can adopt a centrifuge tube with the volume of 1.5 mL; the separation column and the collecting pipe are concentrically fixed through the adapter, the adapter is a collecting pipe cover with a conical through hole in the center and matched with the collecting pipe, the conical through hole is matched with the separation column, so that the separation column and the collecting pipe are concentrically fixed, one end of the separation column is exposed out of the adapter, and the other end of the separation column is positioned inside the collecting pipe. Preferably, the adapter has a vent for equalizing the air pressure inside and outside the collection tube.
Preferably, the desalting washing operation and the eluting operation are performed in a column passing step, the peptide fragment desalting device is adopted, and centrifugal force is adopted to accelerate liquid to pass through the column, and preferably 1000g-3000g centrifugal force is adopted for 1-3min.
Preferably, the protein gel sample mass spectrometry pretreatment method comprises the following steps of (1) carrying out protein reduction treatment and protein alkylation treatment simultaneously:
adding reductive alkylation reaction liquid with the gel volume of 1 to 3 times, and incubating for 20 to 60 minutes at the temperature of 50 to 95 ℃; the reductive alkylation reaction solution contains a reducing agent tri (2-carboxyethyl) phosphine (TCEP) and an alkylating agent Chloroacetamide (CAA), wherein the TCEP concentration is between 5 and 30mM, the CAA concentration is between 20 and 100mM, and preferably, the reductive alkylation reaction solution with the volume of 2 times of the gel block is added to the gel block, and the gel block is incubated for 30min at 56 ℃, and the reductive alkylation reaction solution contains an aqueous solution of 10mM TCEP and 40mM CAA.
According to another aspect of the present invention, there is provided a mass spectrometry pretreatment kit for a protein gel sample, comprising: peptide segment extract, desalting cleaning liquid and desalting eluent;
the peptide extraction liquid is an acidic acetonitrile solution, the acetonitrile concentration is 50-80%, and the acetonitrile concentration is preferably 60%; contains 3 to 10% formic acid, preferably 5% formic acid;
the desalting cleaning solution is 0.1% -0.5% trifluoroacetic acid for desalting and washing, and is preferably 0.2% trifluoroacetic acid solution;
the desalting eluent is alkaline acetonitrile solution, the acetonitrile concentration is between 60% and 90%, and the acetonitrile concentration is preferably 80%; contains 1% -3% NH 4 OH，NH 4 The OH concentration is preferably 1.25%;
the volume ratio of the peptide segment extracting solution to the desalting cleaning solution to the desalting eluent is as follows: 1:0.5-1.5:0.05-0.2.
Preferably, the mass spectrometry pretreatment kit for protein gel samples further comprises: a reductive alkylation reaction solution, wherein the volume ratio of the reductive alkylation reaction solution to the peptide fragment extraction solution is 0.2-0.5:1; the reductive alkylation reaction solution contains a reducing agent tri (2-carboxyethyl) phosphine (TCEP) and an alkylating agent Chloroacetamide (CAA), wherein the TCEP concentration is 5-30mM, and the CAA concentration is 20-100mM.
Preferably, the mass spectrometry pretreatment kit for protein gel samples further comprises: acetonitrile aqueous solution, pancreatin diluent, deionized water, acetonitrile and trypsin; the volume ratio of the acetonitrile aqueous solution, the pancreatin diluent, the deionized water, the acetonitrile and the peptide fragment extract is as follows: 5-20:0.3-0.9:2-4:4-8:1;
the acetonitrile aqueous solution is 100mM NH 4 HCO 3 50% acetonitrile solution;
the pancreatin diluent has a pH of 7.8-8.5 and 50mM NH 4 HCO 3 The solution is used for dissolving trypsin and preparing an enzymolysis reaction solution with the concentration of trypsin enzyme of 0.005-0.02 mug/mu L.
Preferably, the mass spectrometry pretreatment kit for protein gel samples further comprises: the peptide fragment desalting device consists of a detachable nested separation column, an adapter and a collecting pipe. The separation column is a conical tube with two open ends, and the position 3 to 8mm away from the lower end is filled with reversed phase chromatographic packing; the collecting pipe can adopt a centrifuge tube with the volume of 1.5 mL; the separation column and the collecting pipe are concentrically fixed through the adapter, the adapter is a collecting pipe cover with a conical through hole in the center and matched with the collecting pipe, the conical through hole is matched with the separation column, so that the separation column and the collecting pipe are concentrically fixed, one end of the separation column is exposed out of the adapter, and the other end of the separation column is positioned inside the collecting pipe. Preferably, the adapter has a vent for equalizing the air pressure inside and outside the collection tube. The reversed-phase chromatographic packing has a cation exchange effect, when the pH value of a solution is acidic, a peptide segment has positive charges so as to be firmly adsorbed with the reversed-phase chromatographic packing, after desalination and washing are carried out, the pH value of the environment is changed through eluent so that the peptide segment does not have positive charges any more, and therefore the peptide segment is dissolved in alkaline eluent to be eluted, and the preferred reversed-phase chromatographic packing is SDB-RPS reversed-phase chromatographic packing, and is a styrene-divinylbenzene copolymer modified by sulfonic groups.
In general, the above technical solutions conceived by the present invention, compared with the prior art, enable the following beneficial effects to be obtained:
according to the mass spectrum detection pretreatment method for the protein gel sample, provided by the invention, as the cation exchange type reversed phase chromatographic packing is adopted and is matched with the acidic peptide fragment extract and the alkaline peptide fragment eluent, the adsorption effect of the peptide fragment and the reversed phase chromatographic packing is effectively controlled, the solvent replacement process in the sample preparation process is avoided, the solvent is not required to be pumped by a vacuum centrifugal concentrator for solvent replacement, the requirement on experimental conditions is reduced, and the sample preparation time is shortened to about half. Meanwhile, as the adsorption of the peptide segment and the reversed phase chromatographic packing can be effectively controlled, the more peptide segment is reserved, the sensitivity of mass spectrum identification is effectively improved, and the coverage rate and the identification number are obviously improved.
Preferably, the pretreatment time of the further protein mass spectrum detection sample is shortened to 7-10 hours in combination with the simultaneous reduction and alkylation treatment.
Drawings
FIG. 1 is a block diagram of a peptide fragment desalting device. Wherein 1 is a separation column, 2 is an adapter, 3 is a collecting pipe, 4 is a reversed phase chromatographic packing, and 5 is a gas pressure balance hole;
FIG. 2 shows the results of mass spectrometry after treatment of HeLa cell protein gel samples by two methods.
FIG. 3 shows comparison of the identified amounts of proteins detected by mass spectrometry after treatment of gel samples of HeLa cell proteins.
FIG. 4 shows a secondary spectrum of mass spectrometry detection after BSA protein gel sample treatment. The spectrum is evidence for the identification of a peptide fragment of sequence KVPQVSTPTLVEVSR in the BSA protein.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The invention provides a mass spectrum detection pretreatment method of a protein gel sample, which comprises the following steps:
(1) Enzymolysis in the glue: after the lane part to be detected of protein gel electrophoresis is finely crushed, washing, protein reduction treatment, protein alkylation treatment and protease enzymolysis treatment are carried out, and then acidic acetonitrile solution is adopted to extract peptide fragments, so that the peptide fragments have positive charges, and a peptide fragment-acidic acetonitrile solution is obtained; the acidic acetonitrile solution contains 3 to 10% formic acid, preferably 5% formic acid, and the acetonitrile concentration is between 50% and 80%, preferably 60%.
The washing step specifically comprises the following steps: washing and dehydrating the lane part to be detected of the finely crushed protein gel electrophoresis with water, acetonitrile water solution and pure acetonitrile in sequence, wherein the liquid added each time is 3 to 10 times of the gel volume, and preferably 5 times of the gel volume is added for washing; the acetonitrile aqueous solution is preferably 100mM NH 4 HCO 3 The number of times of washing with the acetonitrile aqueous solution is 2-5. The protein gel fine crushing can be realized by a surgical knife cutting mode or byThe glass rod is finished in a grinding and crushing mode.
The protein reduction treatment and the protein alkylation treatment may be performed by sequentially using Dithiothreitol (DTT) and Iodoacetamide (IAA); the protein reduction and alkylation treatments are preferably carried out simultaneously according to the following method:
adding reductive alkylation reaction liquid with the gel volume of 1 to 3 times, and incubating for 20 to 60 minutes at the temperature of 50 to 95 ℃; the reductive alkylation reaction solution contains a reducing agent tri (2-carboxyethyl) phosphine (TCEP) and an alkylating agent Chloroacetamide (CAA), wherein the TCEP concentration is between 5 and 30mM, the CAA concentration is between 20 and 100mM, and preferably, the reductive alkylation reaction solution with the volume of 2 times of the gel block is added to the gel block, and the gel block is incubated for 30min at 56 ℃, and the reductive alkylation reaction solution contains an aqueous solution of 10mM TCEP and 40mM CAA.
The protease enzymolysis treatment can be carried out by trypsin, and specifically comprises the following steps: adding an enzymolysis reaction solution containing 0.005-0.02 μg/μl pancreatin with pH value of 7.8-8.5 and equivalent to gel volume, and reacting at 37deg.C for 3-16 hr.
The extraction operation of the peptide segment by adopting the acidic acetonitrile solution comprises the following steps: adding acidic acetonitrile solution with gel volume of 2 times or more, and 0.3-0.6W/cm 2 And (3) carrying out power density ultrasonic incubation for 5-15 minutes, and collecting a liquid phase after solid-liquid separation. Preferably repeating the extraction operation for 2 times and combining the liquid phases collected by the two extraction operations to obtain the peptide fragment-acidic acetonitrile solution
(2) Desalting the peptide: transferring the peptide-acetonitrile solution obtained in the step (1) into a separation column filled with reverse phase chromatographic packing with cation exchange function, desalting and washing by using trifluoroacetic acid with the mass fraction of 0.1% -0.5%, preferably 0.2%, eluting by using alkaline acetonitrile solution, and collecting eluent containing the peptide as a mass spectrum detection sample.
The reversed-phase chromatographic packing has a cation exchange effect, when the pH value of a solution is acidic, a peptide segment has positive charges so as to be firmly adsorbed with the reversed-phase chromatographic packing, after desalination and washing are carried out, the pH value of the environment is changed through eluent so that the peptide segment does not have positive charges any more, and therefore the peptide segment is dissolved in alkaline eluent to be eluted, and the preferred reversed-phase chromatographic packing is SDB-RPS reversed-phase chromatographic packing, and is a styrene-divinylbenzene copolymer modified by sulfonic groups.
The separation column may be included in a peptide fragment desalting device. The peptide fragment desalting device comprises a detachable nested separation column, an adapter and a collecting pipe. The separation column is a conical tube with two open ends, and the position 3 to 8mm away from the lower end is filled with reversed phase chromatographic packing; the collecting pipe can adopt a centrifuge tube with the volume of 1.5 mL; the separation column and the collecting pipe are concentrically fixed through the adapter, the adapter is a collecting pipe cover with a conical through hole in the center and matched with the collecting pipe, the conical through hole is matched with the separation column, so that the separation column and the collecting pipe are concentrically fixed, one end of the separation column is exposed out of the adapter, and the other end of the separation column is positioned inside the collecting pipe. Preferably, the adapter has a vent for equalizing the air pressure inside and outside the collection tube.
The alkaline acetonitrile solution contains 1% -3% of NH 4 OH, preferably NH 1.25% 4 OH, preferably acetonitrile, at a concentration of between 60% and 90%, preferably 80% acetonitrile;
the operation of desalting and washing by trifluoroacetic acid is specifically as follows: adding 120-180 mu L of solution containing 0.1% -0.5% trifluoroacetic acid into a separation column for adsorbing peptide fragments to be desalted, and passing through the column to obtain a liquid; preferably, the washing operation is repeated 1 to 3 times;
the operation of eluting with basic acetonitrile solution is specifically as follows: adding 15-20 mu L of basic acetonitrile solution to a separation column adsorbed with desalted peptide fragments, and collecting a liquid phase after passing through the column; preferably the elution operation is repeated 2 to 3 times.
Preferably, the desalting washing operation and the eluting operation are performed in a column passing step, a separation column consisting of the detachable nested inner column and the collecting pipe is adopted, and centrifugal force is adopted to accelerate liquid to pass through the column, and preferably 1000g-3000g centrifugal force is adopted for 1-3min.
Compared with the prior art adopting C8 and C18 chromatographic packing which is weak in polarity and adsorbs peptide fragments by steric hindrance, the reversed phase chromatographic packing adopting the cation exchange principle can control and change the adsorption capacity between the packing and the peptide fragments by controlling the pH values of the extracting solution and the eluent, and particularly, the reversed phase chromatographic packing adopting the cation exchange effect can adsorb the peptide fragments dissolved in acidic acetonitrile solution and can carry out peptide fragment elution by alkaline acetonitrile solution, so that the desalting step can be completed without solvent replacement and solvent pumping by a vacuum centrifuge, the requirements on laboratory conditions are greatly reduced, the operation steps are simplified, and the original pretreatment time is shortened to 8-12 hours from 20-30 hours.
Preferably, the pretreatment time of the further protein mass spectrum detection sample is shortened to 7-10 hours in combination with the simultaneous reduction and alkylation treatment.
In addition, we have found that with the pretreatment method of the present invention, the protein gel sample obtains a greater number of peptide identifications and a more complete protein sequence identification coverage during mass spectrometry detection due to the retention of more species of peptide fragments.
The invention provides a pretreatment kit for mass spectrometry detection of protein gel samples, which comprises the following components: peptide segment extract, desalting cleaning liquid and desalting eluent;
the peptide extraction liquid is an acidic acetonitrile solution, and the acetonitrile concentration is 50-80%, preferably 60%; contains 3 to 10% formic acid, preferably 5% formic acid;
the desalting cleaning solution is 0.1% -0.5% trifluoroacetic acid for desalting and washing, and is preferably 0.2% trifluoroacetic acid solution;
the desalting eluent is alkaline acetonitrile solution, and the acetonitrile concentration is between 60% and 90%, preferably 80%; contains 1% -3% NH 4 OH，NH 4 The OH concentration is preferably 1.25%;
the volume ratio of the peptide segment extracting solution to the desalting cleaning solution to the desalting eluent is as follows: 1:0.5-1.5:0.05-0.2.
Preferably, the pretreatment kit for mass spectrometry detection of protein gel sample further comprises: a reductive alkylation reaction solution, wherein the volume ratio of the reductive alkylation reaction solution to the peptide fragment extraction solution is 0.2-0.5:1; the reductive alkylation reaction solution contains a reducing agent tri (2-carboxyethyl) phosphine (TCEP) and an alkylating agent Chloroacetamide (CAA), wherein the TCEP concentration is 5-30mM, and the CAA concentration is 20-100mM.
Preferably, the pretreatment kit for mass spectrometry detection of protein gel sample further comprises: acetonitrile aqueous solution, enzymolysis reaction solution, pancreatin diluent, deionized water, acetonitrile and trypsin; the volume ratio of the acetonitrile aqueous solution, the pancreatin diluent, the deionized water, the acetonitrile and the peptide fragment extract is as follows: 5-20:0.3-0.9:2-4:4-8:1;
the acetonitrile aqueous solution is 100mM NH 4 HCO 3 50% acetonitrile solution;
the pancreatin diluent is 50mM NH4HCO3 solution with the pH value of 7.8-8.5, and is used for dissolving trypsin and preparing enzymolysis reaction liquid with the trypsin concentration of 0.005-0.02 mug/mu L.
Preferably, the mass spectrometry pretreatment kit for protein gel samples further comprises: the peptide fragment desalting device consists of a detachable nested separation column, an adapter and a collecting pipe. The separation column is a conical tube with two open ends, and the position 3 to 8mm away from the lower end is filled with reversed phase chromatographic packing; the collecting pipe can adopt a centrifuge tube with the volume of 1.5 mL; the separation column and the collecting pipe are concentrically fixed through the adapter, the adapter is a collecting pipe cover with a conical through hole in the center and matched with the collecting pipe, the conical through hole is matched with the separation column, so that the separation column and the collecting pipe are concentrically fixed, one end of the separation column is exposed out of the adapter, and the other end of the separation column is positioned inside the collecting pipe. Preferably, the adapter has a vent for equalizing the air pressure inside and outside the collection tube. A separation column packed with charge-adsorbing reverse phase chromatography packing. The reversed-phase chromatographic packing has a cation exchange effect, when the pH value of a solution is acidic, a peptide segment has positive charges so as to be firmly adsorbed with the reversed-phase chromatographic packing, after desalination and washing are carried out, the pH value of the environment is changed through eluent so that the peptide segment does not have positive charges any more, and therefore the peptide segment is dissolved in alkaline eluent to be eluted, and the preferred reversed-phase chromatographic packing is SDB-RPS reversed-phase chromatographic packing, and is a styrene-divinylbenzene copolymer modified by sulfonic groups. Peptide segment desalting device.
The following are examples:
example 1
3 Hela cell extract protein samples were pre-treated and mass detected by the following steps:
(1) Enzymolysis in the glue:
1. washing: cutting off the gel block to be detected, and cutting into about 1mm 3 Small blocks. 500. Mu.L of deionized water was added thereto, and after washing with shaking for 5 minutes, the supernatant was aspirated.
2. Decoloring: 500. Mu.L of acetonitrile aqueous solution was added, and after washing with shaking for 5 minutes, the supernatant was aspirated and repeated 3 times. The acetonitrile aqueous solution was 50% acetonitrile/100 mM ammonium bicarbonate. 500. Mu.L of acetonitrile was added thereto, and after washing with shaking for 5 minutes, the supernatant was aspirated.
3. Reductive alkylation: 200. Mu.L of the reductive alkylation reaction solution was added thereto, and the reaction was carried out at 56℃for 30 minutes. The reductive alkylation reaction solution had a composition of 10mM tris (2-carboxyethyl) phosphine/40 mM chloroacetamide/50 mM ammonium bicarbonate. 500. Mu.L of acetonitrile was added thereto, and after washing with shaking for 5 minutes, the supernatant was aspirated.
4. Enzymolysis: 100. Mu.L of the enzymatic hydrolysis reaction solution was added thereto, and the reaction was carried out at 37℃for 4 hours. The components of the enzymolysis reaction solution are 0.005 mug/mug pancreatin/50 mM ammonium bicarbonate.
5. Extraction: 200. Mu.L of the peptide extract was added thereto, and the mixture was subjected to ultrasonic extraction for 10 minutes. After repeating once, all extracts were mixed. The extract had a composition of 60% acetonitrile/5% formic acid.
(2) Desalting the peptide:
6. desalting: and directly adding the mixed peptide fragment extract into a separation column of a peptide fragment desalting device, and directly placing into a centrifugal machine for centrifugation at 2000g for 2 minutes. The waste liquid enters a waste liquid pipe. 100. Mu.L of desalted washing was added to the column and centrifuged at 2000g for 2 minutes. The waste liquid enters a waste liquid pipe. Repeated 3 times.
The peptide segment desalting device is shown in figure 1 and comprises a detachable nested separation column, an adapter and a collecting pipe, wherein the separation column is a conical pipe with two open ends, and a position 5mm away from the lower end is filled with reversed phase chromatographic packing; the collecting pipe can adopt a centrifuge tube with the volume of 1.5 mL; the inner separation column and the collecting pipe are concentrically fixed through the adapter, the adapter is a collecting pipe cover with a conical through hole in the center and matched with the collecting pipe, the conical through hole is matched with the separation column, so that the separation column and the collecting pipe are concentrically fixed, one end of the separation column of the collecting pipe is exposed out of the adapter, and the other end of the separation column of the collecting pipe is positioned inside the collecting pipe. The adapter has a vent for equalizing the air pressure inside and outside the collection tube. The reversed phase chromatographic packing is SDB-RPS reversed phase chromatographic packing.
7. Eluting: 20. Mu.L of desalted eluent was added to the column and centrifuged at 2000g for 1 min. Collecting the eluent in a sample collection tube. The desalted eluate containing the peptide fragments was mixed 3 times for subsequent mass spectrometry detection. The desalting eluent comprises 1.25% ammonia water/80% acetonitrile.
The same triplicate HeLa cell protein gel samples were treated with the existing in-gel enzymatic and desalting methods (see Sample preparation by in-gel digestion for mass spectrometry-based proteomics. Animal biological Chem (2007) 389:991-1002.DOI10.1007/s 00216-007-1451-4) and mass spectrometry and data analysis were performed. The results of the protein and peptide fragment identification are shown in the following table.
The details of the amounts of proteins identified by the two methods are given in the following table.
Comparing the amounts of proteins identified by the two methods, as shown in FIG. 2, the method provided by the invention can cover 89.68% of the protein identifications of the prior methods, and 62.73% of the novel protein identifications are also shown in FIG. 3. That is, in this example, the method of the present invention increased the number of protein identifications by 52.41% compared to the existing method.
Compared with the prior art, the proteogel enzymolysis and desalting method provided by the invention has the advantages that the operation steps are reduced, the treatment time is shortened, the experimental cost is reduced, the mass spectrum identification effect of gel protein samples containing complex protein components can be effectively improved, and more protein and peptide fragment identification quantity is obtained, so that the proteogel enzymolysis and desalting method is a more excellent proteogel enzymolysis and desalting method.
Example 2
2 samples of Bovine Serum Albumin (BSA) were treated and mass detected by the following procedure.
The specific processing steps are as follows:
(1) Enzymolysis in the glue:
1. washing: cutting off the gel block to be detected, and cutting into about 1mm 3 Small blocks. 500. Mu.L of deionized water was added thereto, and after washing with shaking for 5 minutes, the supernatant was aspirated.
2. Decoloring: 500. Mu.L of acetonitrile aqueous solution was added, and after washing with shaking for 5 minutes, the supernatant was aspirated and repeated 3 times. The acetonitrile aqueous solution was 50% acetonitrile/100 mM ammonium bicarbonate. 500. Mu.L of acetonitrile was added thereto, and after washing with shaking for 5 minutes, the supernatant was aspirated.
3. Reductive alkylation: 200. Mu.L of the reductive alkylation reaction solution was added thereto, and the reaction was carried out at 56℃for 30 minutes. The reductive alkylation reaction solution had a composition of 10mM tris (2-carboxyethyl) phosphine/40 mM chloroacetamide/50 mM ammonium bicarbonate. 500. Mu.L of acetonitrile was added thereto, and after washing with shaking for 5 minutes, the supernatant was aspirated.
4. Enzymolysis: 100. Mu.L of the enzymatic hydrolysis reaction solution was added thereto, and the reaction was carried out at 37℃for 4 hours. The components of the enzymolysis reaction solution are 0.005 mug/mug pancreatin/50 mM ammonium bicarbonate.
5. Extraction: 200. Mu.L of the peptide extract was added thereto, and the mixture was subjected to ultrasonic extraction for 10 minutes. After repeating once, all extracts were mixed. The extract had a composition of 60% acetonitrile/5% formic acid.
(2) Desalting the peptide:
6. desalting: the mixed peptide extract was directly added to the separation column of the peptide desalting device described in example 1, and the mixture was directly placed in a centrifuge for centrifugation at 2000g for 2 minutes. The waste liquid enters a waste liquid pipe. 100. Mu.L of desalted washing was added to the column, and the column was centrifuged at 2000g for 2 minutes. The waste liquid enters a waste liquid pipe. Repeated 3 times.
7. Eluting: 20. Mu.L of desalted eluent was added to the desalter column and centrifuged at 2000g for 1 min. Collecting the eluent in a sample collection tube. The desalted eluate containing the peptide fragments was mixed 3 times for subsequent mass spectrometry detection. The desalting eluent comprises 1.25% ammonia water/80% acetonitrile.
The same 2 BSA protein gel samples were treated by the existing in-gel enzymolysis and desalting methods, and mass spectrometry and data analysis were performed. The mass spectrum test results are shown in fig. 4, and the identification results are shown in the following table.
Compared with the prior art, the proteolysis and desalting method provided by the invention has the advantages that the operation steps are reduced, the treatment time is shortened, the experimental cost is reduced, the mass spectrum identification effect of the gel protein sample containing single protein component can be effectively improved, and more peptide fragment identification quantity and more complete protein sequence identification coverage rate are obtained, so that the proteolysis and desalting method is a more excellent proteolysis and desalting method in albumin glue.
Example 3
5 samples of Bovine Serum Albumin (BSA) were treated and mass detected by the following procedure. The 5 protein samples contained 10. Mu.g, 1. Mu.g, 0.1. Mu.g, 0.01. Mu.g and 0.001. Mu.g BSA protein, respectively.
The specific processing steps are as follows:
(1) Enzymolysis in the glue:
1. washing: cutting off the gel block to be detected, and cutting into about 1mm 3 Small blocks. 500. Mu.L of deionized water was added thereto, and after washing with shaking for 5 minutes, the supernatant was aspirated.
2. Decoloring: 500. Mu.L of acetonitrile aqueous solution was added, and after washing with shaking for 5 minutes, the supernatant was aspirated and repeated 3 times. The acetonitrile aqueous solution was 50% acetonitrile/100 mM ammonium bicarbonate. 500. Mu.L of acetonitrile was added thereto, and after washing with shaking for 5 minutes, the supernatant was aspirated.
3. Reductive alkylation: 200. Mu.L of the reductive alkylation reaction solution was added thereto, and the reaction was carried out at 56℃for 30 minutes. The reductive alkylation reaction solution had a composition of 10mM tris (2-carboxyethyl) phosphine/40 mM chloroacetamide/50 mM ammonium bicarbonate. 500. Mu.L of acetonitrile was added thereto, and after washing with shaking for 5 minutes, the supernatant was aspirated.
4. Enzymolysis: 100. Mu.L of the enzymatic hydrolysis reaction solution was added thereto, and the reaction was carried out at 37℃for 4 hours. The components of the enzymolysis reaction solution are 0.005 mug/mug pancreatin/50 mM ammonium bicarbonate.
5. Extraction: 200. Mu.L of the peptide extract was added thereto, and the mixture was subjected to ultrasonic extraction for 10 minutes. After repeating once, all extracts were mixed. The extract had a composition of 60% acetonitrile/5% formic acid.
(2) Desalting the peptide:
6. desalting: the mixed peptide extract was directly added to the separation column of the peptide desalting device described in example 1, and the mixture was directly placed in a centrifuge for centrifugation at 2000g for 2 minutes. The waste liquid enters a waste liquid pipe. 100. Mu.L of desalted washing was added to the column and centrifuged at 2000g for 2 minutes. The waste liquid enters a waste liquid pipe. Repeated 3 times.
7. Eluting: 20. Mu.L of desalted eluent was added to the desalter column and centrifuged at 2000g for 1 min. Collecting the eluent in a sample collection tube. The desalted eluate containing the peptide fragments was mixed 3 times for subsequent mass spectrometry detection. The desalting eluent comprises 1.25% ammonia water/80% acetonitrile.
After each sample was detected by mass spectrometry, the identification results of the BSA protein are shown in the following table:
| sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | |
| BSA protein amount | 10μg | 1μg | 0.1μg | 0.01μg | 0.001μg |
| BSA mass spectrometry | Is that | Is that | Is that | Is that | Whether or not |
| Identification number of peptide fragment | 99 | 50 | 11 | 4 | 0 |
| Sequence coverage | 85.17％ | 59.14％ | 19.1％ | 7.12％ | 0 |
The embodiment shows that the pretreatment method for the protein gel sample can realize mass spectrum detection and identification of the protein gel with the content as low as 0.01 mu g. The pretreatment method provided by the invention can be effectively used for mass spectrum detection and identification of trace proteins in protein gel.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (13)
1. The mass spectrometry pretreatment method for the protein gel sample is characterized by comprising the following steps of:
(1) Enzymolysis in the glue: after the lane part to be detected of protein gel electrophoresis is finely crushed, washing, protein reduction treatment, protein alkylation treatment and protease enzymolysis treatment are carried out, and then acidic acetonitrile solution is adopted to carry out peptide segment extraction, so as to obtain peptide segment-acidic acetonitrile solution; the acidic acetonitrile solution contains 3 to 10 percent of formic acid, and the acetonitrile concentration is between 50 and 80 percent;
(2) Desalting the peptide: transferring the peptide-acetonitrile solution obtained in the step (1) to a separation column filled with reversed phase chromatographic packing with cation exchange function, desalting and washing by using trifluoroacetic acid solution with mass fraction of 0.1% -0.5%, eluting by using alkaline acetonitrile solution, and collecting eluent containing peptide as a mass spectrum detection sample; the reversed phase chromatographic packing is SDB-RPS reversed phase chromatographic packing; the alkaline acetonitrile solution contains 1% -3% of NH 4 OH; acetonitrile concentration between 60% and 90%;
the reversed phase chromatographic packing has cation exchange function, when the pH value of the solution is acidic, the peptide segment has positive charges so as to be firmly adsorbed with the reversed phase chromatographic packing, and after desalination and washing, the pH value of the environment is changed through eluent, so that the peptide segment does not have positive charges any more, and is dissolved in alkaline eluent to be eluted.
2. The method for mass spectrometry pretreatment of protein gel samples according to claim 1, wherein the separation column is contained in a peptide fragment desalting device; the peptide fragment desalting device consists of a detachable nested separation column, an adapter and a collecting pipe; the separation column is a conical tube with two open ends, and the position 3 to 8mm away from the lower end is filled with reversed phase chromatographic packing; the collecting pipe adopts a centrifuge tube of 1.5 mL; the separation column and the collecting pipe are concentrically fixed through the adapter, the adapter is a collecting pipe cover with a conical through hole in the center and matched with the collecting pipe, the conical through hole is matched with the separation column, so that the separation column and the collecting pipe are concentrically fixed, one end of the separation column is exposed out of the adapter, and the other end of the separation column is positioned inside the collecting pipe.
3. The method for mass spectrometry pretreatment of protein gel samples according to claim 2, wherein the adapter has a vent hole for balancing the air pressure inside and outside the collection tube.
4. The method for pretreatment of mass spectrometry detection of protein gel sample according to claim 1, wherein the desalting washing with trifluoroacetic acid is specifically: adding 120-180 mu L of trifluoroacetic acid solution containing 0.1% -0.5% into a separation column for adsorbing peptide fragments to be desalted, and passing through the column to obtain a liquid; the washing operation is repeated 1 to 3 times;
the operation of eluting with basic acetonitrile solution is specifically as follows: adding 15-20 mu L of basic acetonitrile solution to a separation column adsorbed with desalted peptide fragments, and collecting a liquid phase after passing through the column; the elution operation was repeated 2 to 3 times.
5. A method of mass spectrometry pretreatment of protein gel samples according to claim 2 or 3, wherein the desalting washing and eluting steps are performed by using a peptide desalting device comprising the detachably nested separation column, adapter and collection tube, accelerating liquid flow through the column by using centrifugal force, and centrifuging for 1-3min by using 1000g-3000 g.
6. The method for pretreatment for mass spectrometry detection of protein gel samples according to claim 1, wherein the protein reduction treatment and the protein alkylation treatment in step (1) are carried out simultaneously according to the following method:
adding reductive alkylation reaction liquid with the gel volume of 1 to 3 times, and incubating for 20 to 60 minutes at the temperature of 50 to 95 ℃; the reductive alkylation reaction solution contains a reducing agent tri (2-carboxyethyl) phosphine and an alkylating agent chloroacetamide, wherein the TCEP concentration is between 5 and 30 and mM, and the CAA concentration is between 20 and 100mM.
7. The method for mass spectrometry pretreatment of protein gel samples according to claim 6, wherein a 2-fold gel volume of a reductive alkylation reaction solution containing 10mM TCEP and 40mM CAA in water is added to the gel block and incubated at 56℃for 30 min.
8. A mass spectrometry pretreatment kit for a protein gel sample, comprising: a peptide segment extracting solution, a desalting cleaning solution, a desalting eluent and a separation column with reverse phase chromatographic packing with cation exchange function;
the peptide extraction liquid is an acidic acetonitrile solution, and the acetonitrile concentration is between 50% and 80%; contains 3 to 10% formic acid;
the desalting cleaning solution is prepared by desalting and washing with trifluoroacetic acid accounting for 0.1% -0.5%;
the desalting eluent is alkaline acetonitrile solution, and the acetonitrile concentration is between 60% and 90%; contains 1% -3% NH 4 OH；
The volume ratio of the peptide segment extracting solution to the desalting cleaning solution to the desalting eluent is as follows: 1:0.5-1.5:0.05-0.2;
the reversed phase chromatographic packing is SDB-RPS reversed phase chromatographic packing.
9. The pretreatment kit for mass spectrometry detection of protein gel samples according to claim 8, wherein the peptide fragment extract has an acetonitrile concentration of 60% and a formic acid concentration of 5%;
the desalted cleaning liquid is 0.2% trifluoroacetic acid solution;
the concentration of acetonitrile in the desalted eluent is 80%, and the concentration of NH is 4 The OH concentration was 1.25%.
10. The pretreatment kit for mass spectrometry detection of protein gel samples of claim 8, further comprising: a reductive alkylation reaction solution, wherein the volume ratio of the reductive alkylation reaction solution to the peptide fragment extraction solution is 0.2-0.5:1; the reductive alkylation reaction solution contains a reducing agent tri (2-carboxyethyl) phosphine and an alkylating agent chloroacetamide, wherein the TCEP concentration is 5-30mM, and the CAA concentration is 20-100mM.
11. The pretreatment kit for mass spectrometry detection of protein gel samples of claim 8, further comprising: acetonitrile aqueous solution, pancreatin diluent, deionized water, acetonitrile and trypsin; the volume ratio of the acetonitrile aqueous solution, the pancreatin diluent, the deionized water, the acetonitrile and the peptide fragment extract is as follows: 5-20:0.3-0.9:2-4:4-8:1;
the acetonitrile aqueous solution is 100mM NH 4 HCO 3 50% acetonitrile solution;
the pancreatin diluent has a pH of 7.8-8.5 and 50mM NH 4 HCO 3 The solution is used for dissolving trypsin and preparing an enzymolysis reaction solution with the concentration of trypsin enzyme of 0.005-0.02 mug/mu L.
12. The pretreatment kit for mass spectrometry detection of protein gel samples of claim 8, further comprising: the peptide segment desalting device consists of a detachable nested separation column, an adapter and a collecting pipe; the separation column is a conical tube with two open ends, and the position 3 to 8mm away from the lower end is filled with reversed phase chromatographic packing; the collecting pipe adopts a centrifuge tube of 1.5 mL; the separation column and the collecting pipe are concentrically fixed through the adapter, the adapter is a collecting pipe cover with a conical through hole in the center and matched with the collecting pipe, the conical through hole is matched with the separation column, so that the separation column and the collecting pipe are concentrically fixed, one end of the separation column is exposed out of the adapter, and the other end of the separation column is positioned inside the collecting pipe.
13. The pretreatment kit for mass spectrometry of protein gel samples according to claim 12, wherein the adapter has a vent for balancing the air pressure inside and outside the collection tube; when the pH value of the solution is acidic, the peptide fragment is positively charged so as to be firmly adsorbed with the reversed phase chromatographic packing, and after desalination and washing are carried out, the pH value of the environment is changed through eluent, so that the peptide fragment is not positively charged any more, and is dissolved in alkaline eluent to be eluted.
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