Method for detecting serum proteins by capillary electrophoresis tandem mass spectrometry

Abstract

The application provides a method for detecting serum proteins by capillary electrophoresis tandem mass spectrometry, and belongs to the technical field of biochemical detection. The human serum is directly mixed with a buffer, and then subjected to on-line analysis of capillary electrophoresis and mass spectrometry. The effective separation of high-abundance proteins in the serum is realized by capillary electrophoresis separation. The on-line analysis technology of capillary electrophoresis and mass spectrometry is used, each protein is ionized by a mass spectrometry ion source for mass spectrometry analysis, the sample demand can be effectively reduced, the sample pretreatment difficulty can be reduced, the on-line separation degree of the protein can be increased, the number and the type of the serum proteins can be accurately identified, the efficient separation and the high-sensitivity detection of the human serum sample can be realized, the efficient and high-accuracy identification of the human serum proteins is realized, and a basis is provided for further carrying out the application research work of serum protein electrophoresis in the clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biochemical detection, in particular to a method for detecting serum proteins by capillary electrophoresis tandem mass spectrometry. BACKGROUND

[0002] Serum protein electrophoresis is a common method for identifying and quantitatively screening variants of proteins (albumin, alpha-protein, beta-protein and M protein) in serum, especially the most characteristic changes in patients with kidney disease (such as nephrotic syndrome, monoclonal immunoglobulin-related kidney damage), cirrhosis and plasma cell disease (such as multiple myeloma), which is of great significance in clinical diagnosis. The current electrophoresis method for detecting serum proteins includes cellulose acetate film electrophoresis (CAE), agarose gel electrophoresis (AGE) and capillary electrophoresis (CE). Capillary electrophoresis has become a routine separation method for clinical analysis of serum proteins due to its high separation efficiency of serum proteins. The commonly used detection method for serum protein electrophoresis is capillary electrophoresis combined with ultraviolet detection, which can only obtain the ultraviolet absorption peak of the protein, but cannot identify each protein online. Moreover, the ultraviolet detector has low sensitivity, low accuracy and high sample requirement. The liquid chromatography-mass spectrometry method is a combined technique that combines liquid chromatography separation technology with accurate protein molecular mass detection technology of mass spectrometry. Currently, serum proteins can also be separated and accurately identified, but liquid chromatography has poor separation effect on serum proteins, and requires complex sample pretreatment such as affinity immunoprecipitation and solid-phase extraction, resulting in large sample requirement, high detection cost, high error rate and low efficiency due to complex processing steps. SUMMARY

[0003] The present application aims to provide a method for detecting serum proteins by capillary electrophoresis tandem mass spectrometry, which can realize efficient separation and high-accuracy identification of human serum proteins, and has simple sample processing.

[0004] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0005] The present application provides a method for detecting serum proteins by capillary electrophoresis tandem mass spectrometry, comprising the following steps:

[0006] Mixing the serum sample and the buffer to obtain a sample liquid to be tested;

[0007] Performing capillary electrophoresis mass spectrometry online analysis and detection on the sample liquid to be tested, and identifying the serum proteins according to the molecular weight obtained by mass spectrometry;

[0008] The conditions of the capillary electrophoresis in the capillary electrophoresis-mass spectrometry online analysis and detection include: the separation buffer is 30wt% acetic acid aqueous solution, the separation voltage is 30kV, the injection mode is pressure injection, the pressure of the pressure injection is 100mbar, and the injection time is 7s;

[0009] The conditions of the mass spectrometry in the capillary electrophoresis-mass spectrometry online analysis and detection include: positive ion scanning mode, the dry gas flow rate is 6L / min, the dry temperature is 380℃, the fragmentation voltage is 365V, the skimmer voltage is 65V, the MS scanning range is m / z 800-3200, and the scanning rate is 1spectrum / s.

[0010] Preferably, the buffer is ammonium acetate solution, and the concentration of the ammonium acetate solution is 100mmol / L.

[0011] Preferably, the solvent of the ammonium acetate solution is 4wt% acetic acid aqueous solution.

[0012] Preferably, the volume ratio of the serum sample to the buffer is 1:19, and the injection amount of the capillary electrophoresis is 40μL.

[0013] Preferably, the separation capillary used in the capillary electrophoresis is a neutral coating capillary with a length of 100cm.

[0014] Preferably, the ion source conditions in the capillary electrophoresis-mass spectrometry online analysis and detection include: an electric osmosis flow driven coaxial sheath flow liquid electro spray ion source, and the sheath flow liquid is a mixed aqueous solution containing 0.5% formic acid and 10% isopropanol by volume fraction.

[0015] Preferably, the ion source conditions in the capillary electrophoresis-mass spectrometry online analysis and detection include: the material of the electro spray needle is borosilicate glass; the spray voltage is 2.2kV, the distance between the electro spray needle and the mass spectrometer is 4.0mm, and the distance between the capillary and the spray needle is 0.67mm.

[0016] Preferably, the outer diameter of the electro spray needle is 1.5mm, the inner diameter is 1.17mm, and the opening diameter is 20μm.

[0017] Preferably, the mass spectrometry in the capillary electrophoresis-mass spectrometry detection is detected by a time-of-flight mass spectrometry system.

[0018] The application provides a method for detecting serum proteins by capillary electrophoresis tandem mass spectrometry, wherein human serum is mixed with a buffer, and then subjected to on-line analysis by capillary electrophoresis and mass spectrometry (the sample is first separated by CE, the separated proteins are ionized and then directly subjected to mass spectrometric analysis and detection), the effective separation of high-abundance proteins (albumin, transferrin and immunoglobulin) in serum is achieved by capillary electrophoresis separation, each protein is ionized by a mass spectrometry ion source for mass spectrometric analysis by using the on-line analysis technology of capillary electrophoresis and mass spectrometry, the sample requirement can be effectively reduced, the sample pretreatment difficulty can be reduced, the on-line separation degree of proteins can be increased, and the number and types of serum proteins can be accurately identified.

[0019] The application combines the high-efficiency separation of capillary electrophoresis and the qualitative ability of high-resolution mass spectrometry, adopts capillary electrophoresis for protein separation, and is superior to traditional gel electrophoresis in separation efficiency; high-resolution mass spectrometry is adopted for protein detection and qualitative identification, which is more sensitive than traditional electrophoresis by coloration, and the qualitative identification of serum proteins is more accurate and reliable.

[0020] Further, the application adopts specific buffer to treat serum samples, and does not need to analyze the composition of serum proteins by traditional electrophoresis, thereby solving the problems of complex operation and insufficient qualitative ability of traditional electrophoresis.

[0021] Compared with the traditional capillary electrophoresis-liquid chromatography mass spectrometry method, the method of the application does not need to prepare specific diluents, is accurate, efficient and low in cost, realizes the effective separation of high-abundance proteins in human serum and the accurate identification of the overall molecular weight level of proteins, and has important significance for promoting the application of high-abundance proteins in human serum in clinical diagnosis and treatment.

[0022] Compared with the method for detecting serum proteins by combining two-dimensional electrophoresis with tandem mass spectrometry (differential comparison is performed by two-dimensional electrophoresis, serum proteins are identified by matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI / TOF / TOF / MS), the steps are numerous, the sample pretreatment is complex, and on-line identification and analysis of serum proteins cannot be achieved), the method of the application can realize on-line identification and analysis of serum proteins, and the sample treatment is simple. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A method flow chart of the present application;

[0024] Figure 2 A total ion chromatogram of capillary electrophoresis mass spectrometry analysis of human serum proteins;

[0025] Figure 3 A mass spectrum of 6 proteins of capillary electrophoresis mass spectrometry analysis of human serum proteins;

[0026] Figure 4 A mass spectrum of 6 proteins of capillary electrophoresis mass spectrometry analysis of human serum proteins. DETAILED DESCRIPTION

[0027] The present application provides a method for detecting serum proteins by capillary electrophoresis tandem mass spectrometry, comprising the following steps:

[0028] Mixing a serum sample and a buffer to obtain a sample liquid to be detected;

[0029] Detecting the sample liquid to be detected by capillary electrophoresis mass spectrometry online analysis, and identifying serum proteins according to the molecular weight obtained by mass spectrometry;

[0030] The conditions of capillary electrophoresis in the capillary electrophoresis mass spectrometry online analysis detection include: a separation buffer of 30wt% acetic acid aqueous solution, a separation voltage of 30kV, a pressure injection mode, a pressure of 100mbar for pressure injection, and an injection time of 7s;

[0031] The conditions of mass spectrometry in the capillary electrophoresis mass spectrometry online analysis detection include: a positive ion scanning mode, a dry gas flow rate of 6L / min, a dry temperature of 380℃, a fragmentation voltage of 365V, a skimmer voltage of 65V, an MS scanning range of m / z 800-3200, and a scanning rate of 1spectrum / s.

[0032] In the present application, if not otherwise specified, the required reagents or equipment are all commercially available goods well known to those skilled in the art.

[0033] The present application mixes a serum sample and a buffer to obtain a sample liquid to be detected.

[0034] The present application does not have special limitations on the source of the serum sample, which can be obtained in a manner well known in the art. In the embodiments of the present application, the serum frozen in a-80℃ refrigerator is preferably used, which is thawed at room temperature until the serum sample is restored to room temperature for preparing the sample liquid to be detected.

[0035] In the present application, the buffer solution is preferably an ammonium acetate solution, the concentration of the ammonium acetate solution is preferably 100 mmol / L, and the solvent used in the ammonium acetate solution is preferably a 4 wt% acetic acid aqueous solution. The preparation process of the buffer solution in the present application is not particularly limited, and the buffer solution with the above-mentioned concentration can be prepared according to the method well known in the art; in the embodiment of the present application, 4 mL of acetic acid, 96 mL of water and 0.77 g of ammonium acetate are mixed to prepare the buffer solution. The buffer solution used in the present application can dilute the sample matrix, so that the protein to be tested can be charged, and the sample can be more compatible with the separation buffer of CE.

[0036] In the present application, the volume ratio of the serum sample to the buffer solution is preferably 1:19.

[0037] In the present application, the mixture of the serum sample and the buffer solution is preferably mixed uniformly by using a vortex instrument for 30 s to obtain a sample liquid to be tested.

[0038] After obtaining the sample liquid to be tested, the present application performs on-line analysis and detection of the sample liquid to be tested by capillary electrophoresis mass spectrometry, and identifies serum proteins according to the molecular weight obtained by mass spectrometry.

[0039] In the present application, the capillary electrophoresis mass spectrometry instrument (CE-MS) used in the on-line analysis and detection of capillary electrophoresis mass spectrometry is the CMP ECE-001 type electrophoresis instrument and the CMP EMAST II type CE-MS ion source disclosed by the CMP Technology Company, a subsidiary of Yongda Zhiyuan Science and Technology Co., Ltd., which has high sensitivity, good separation degree for serum proteins, and can realize the separation and real-time identification of proteins in serum.

[0040] In the present application, the conditions of capillary electrophoresis in the on-line analysis and detection of capillary electrophoresis mass spectrometry include that the separation buffer is a 30 wt% acetic acid aqueous solution, the separation voltage is 30 kV, the injection mode is pressure injection, the pressure of pressure injection is 100 mbar, and the injection time is 7 s.

[0041] In the present application, the separation capillary used in the capillary electrophoresis is preferably a neutral coating capillary with a length of 100 cm; the neutral coating capillary in the present application is not particularly limited, and the corresponding material well known in the art can be used.

[0042] In the present application, the injection amount of the capillary electrophoresis is preferably 40 μL.

[0043] In the on-line analysis and detection of capillary electrophoresis mass spectrometry in the present application, first, electrophoresis separation is performed by capillary electrophoresis, then ionization is performed by the CE-MS ion source, on-line analysis of CE and MS is realized, and sample ionization is further promoted.

[0044] In the present application, the ion source conditions in the on-line analysis and detection of capillary electrophoresis-mass spectrometry preferably include: coaxial sheath flow liquid electro-spray ion source driven by electroosmotic flow, the sheath flow liquid is a mixed aqueous solution containing 0.5% formic acid and 10% isopropanol by volume fraction; the material of the electro-spray needle is borosilicate glass; the spray voltage is 2.2 kV, the distance between the electro-spray needle and the mass spectrometer is 4.0 mm, and the distance between the capillary and the spray needle is 0.67 mm; the outer diameter of the electro-spray needle is 1.5 mm, the inner diameter is 1.17 mm, and the opening diameter is 20 μm.

[0045] In the present application, the conditions of the mass spectrometer in the on-line analysis and detection of capillary electrophoresis-mass spectrometry include: positive ion scanning mode, dry gas flow rate is 6 L / min, dry temperature is 380℃, fragmentation voltage is 365 V, skimmer is 65 V, MS scanning range m / z 800-3200, scanning rate is 1 spectrum / s.

[0046] In the present application, the mass spectrometer in the capillary electrophoresis-mass spectrometry detection preferably applies a time-of-flight mass spectrometry system for detection.

[0047] Figure 1 The flow chart of the method of the present application; the present application adopts capillary electrophoresis-mass spectrometry on-line analysis and detection, separates the serum sample after pretreatment by capillary electrophoresis, then ionizes by mass spectrometry, and then performs mass spectrometry analysis, so as to realize the identification of serum proteins.

[0048] The technical solutions in the present application will be clearly and completely described below in combination with the embodiments in the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0049] In the following embodiments, the preparation method of the buffer is as follows: 4 mL of acetic acid, 96 mL of water and 0.77 g of ammonium acetate are mixed to obtain a buffer with a concentration of 100 mmol / L.

[0050] Example 1

[0051] (1) Pretreatment of the sample to be tested: take out the human serum frozen in the-80℃ refrigerator, thaw at room temperature, take 2 μL of serum sample after the sample is restored to room temperature, then take 38 μL of buffer (4% acetic acid and 100 mmol / L ammonium acetate) for dilution, mix uniformly by using a vortex instrument for 30 s, and wait for sample analysis.

[0052] (2) The analysis conditions of capillary electrophoresis-mass spectrometry are as follows:

[0053] Capillary electrophoresis conditions: the separation capillary was a 100cm neutral coated capillary, the separation buffer was a 30wt% acetic acid aqueous solution, the separation voltage was 30kV, and the sample was injected for 7s at a pressure of 100mbar.

[0054] Ion source conditions for capillary electrophoresis-tandem mass spectrometry: an electroosmotic flow-driven coaxial sheath flow electrospray ion source; the sheath flow was a mixed aqueous solution containing 0.5% formic acid and 10% isopropanol by volume; the electrospray needle was made of borosilicate glass with an outer diameter of 1.5 mm, an inner diameter of 1.17 mm, and an opening diameter of 20 μm; the spray voltage was 2.2 kV; the distance between the spray needle and the mass spectrometer was 4.0 mm; and the distance between the capillary and the spray needle was 0.67 mm.

[0055] Mass spectrometry conditions: Time-of-flight mass spectrometry system was used for detection. Scanning mode: positive ion scan mode, drying gas flow rate of 6 L / min and drying temperature of 380 °C, fragmentation voltage of 365 V, splitter voltage of 65 V, MS scan range of m / z 800~3200, scan rate of 1 spectrum / s;

[0056] The obtained mass spectrometry data is deconvolved to obtain the molecular weight of each protein, and each protein in the serum is identified by precise molecular weight.

[0057] Figure 2 Total ion current chromatogram of human serum protein analysis by capillary electrophoresis-mass spectrometry;

[0058] Figure 3 Mass spectra of six proteins analyzed by capillary electrophoresis-mass spectrometry of human serum proteins;

[0059] Figure 4 This is a precise molecular weight map of six proteins after unconvolution in human serum protein capillary electrophoresis-mass spectrometry analysis.

[0060] Depend on Figures 2 to 4 It was found that a total of 6 serum proteins were identified from human serum proteins, including 3 albumin isoforms (P1, P2, P3), 1 transferrin (p4), 1 glycosylated light chain (p5), and 1 free polypeptide (p6).

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting serum proteins by capillary electrophoresis tandem mass spectrometry, comprising the following steps: mixing a serum sample and a buffer to obtain a sample liquid to be detected; performing capillary electrophoresis mass spectrometry online analysis and detection on the sample liquid to be detected, and identifying serum proteins according to the molecular weight obtained by mass spectrometry; the separation buffer is 30 wt% acetic acid aqueous solution, the separation voltage is 30 kV, the sample injection mode is pressure injection, the pressure of the pressure injection is 100 mbar, and the sample injection time is 7 s; the conditions of mass spectrometry in the capillary electrophoresis mass spectrometry online analysis and detection include: positive ion scanning mode, a dry gas flow rate of 6 L / min, a dry temperature of 380 ℃, a fragmentation voltage of 365 V, a skimmer voltage of 65 V, an MS scanning range of m / z 800-3200, and a scanning rate of 1 spectrum / s; the buffer is an ammonium acetate solution, the concentration of the ammonium acetate solution is 100 mmol / L; and the volume ratio of the serum sample to the buffer is 1:

19. The solvent used in the ammonium acetate solution is 4 wt% acetic acid aqueous solution. The sample injection amount of the capillary electrophoresis is 40 μL. The conditions of capillary electrophoresis in the on-line analysis and detection of capillary electrophoresis mass spectrometry include: The separation capillary used in the capillary electrophoresis is a neutral coating capillary with a length of 100 cm. The ion source conditions in the capillary electrophoresis mass spectrometry online analysis and detection include: an electric osmosis flow driven coaxial sheath flow liquid electrospray ion source, and a sheath flow liquid being a mixed aqueous solution containing 0.5% formic acid and 10% isopropanol by volume fraction. The ion source conditions in the capillary electrophoresis mass spectrometry online analysis and detection include: the material of the electrospray needle is borosilicate glass; the spray voltage is 2.2 kV, the distance between the electrospray needle and the mass spectrometer is 4.0 mm, and the distance between the capillary and the electrospray needle is 0.67 mm. The outer diameter of the electrospray needle is 1.5 mm, the inner diameter is 1.17 mm, and the opening diameter is 20 μm.

2. The method of claim 1, wherein, The mass spectrometry in the capillary electrophoresis-mass spectrometry detection is performed by using a time-of-flight mass spectrometry system.

3. The method according to claim 1 or 2, characterized in that, ​ 4. The method of claim 3, wherein, ​ 5. The method of claim 1, wherein, ​ 6. The method according to claim 1 or 5, characterized in that, ​ 7. The method of claim 6, wherein, ​ 8. The method of claim 1, wherein, ​

Images