Optimized matrix of MALDI (matrix-assisted laser desorption ionization) and preparation method thereof

Abstract

The invention discloses an optimized matrix of MALDI and a preparation method, the optimized matrix of MALDI is a graphene oxide matrix, and the preparation of the matrix comprises the following steps: preparing a graphene oxide dispersion liquid; mixing the graphene oxide dispersion liquid with the auxiliary matrix solution under a dark condition to form a homogeneous suspension liquid; and vertically dropwise adding the mixed solution to a polished stainless steel target plate by adopting a micropipettor, and carrying out vacuum drying in a constant-temperature and constant-humidity box to form a nanoscale composite crystal film layer so as to obtain the graphene oxide substrate. The characteristics of a carbon material and a traditional organic matrix are integrated, and the graphene oxide with higher uniformity, high hydrophilicity and solid-phase adsorption capacity is used for assisting the traditional solid matrix with high ionization capacity, so that a to-be-detected object and the organic matrix are uniformly adsorbed on the graphene oxide, the generation of hot spots is avoided, and the detection accuracy is improved. The matrix is helped to form more uniform crystals, and the reproducibility of MALDI detection is improved.

Description

Technical Field

[0001] The present application belongs to the field of mass spectrometry analysis technology, and specifically relates to an optimized MALDI matrix and a preparation method. Background Art

[0002] Matrix-assisted laser desorption / ionization mass spectrometry (MALDI) is a soft ionization technique characterized by rapid scanning, high throughput, and suitability for the analysis of biomacromolecules. Typically, a MALDI sample is provided on a MALDI plate. A laser selects spots on the plate for ionization. However, MALDI-TOF / MS inherently forms uneven cocrystallizations between the analyte and the matrix, leading to poor reproducibility, increased sample analysis time, and the inability to perform automated sampling. This phenomenon of uneven cocrystallization is known as a "hotspot" or "sweet spot." Summary of the Invention

[0003] The present application provides an optimized MALDI matrix and a preparation method to solve the technical problem that traditional matrices form uneven co-crystallization characteristics resulting in poor reproducibility.

[0004] To solve the above technical problems, the present application adopts a technical solution: a method for preparing an optimized MALDI matrix, comprising:

[0005] Preparation of graphene oxide dispersion: 50-100 mg of graphene oxide powder was weighed and added to 100 mL of ultrapure water. The mixture was magnetically stirred at 25°C and 800 rpm for 15 minutes. The mixture was then ultrasonicated for 30 minutes to obtain a uniform graphene oxide dispersion.

[0006] S2. Matrix mixing: 1 mL of the graphene oxide dispersion was mixed with 5-10 mL of the auxiliary matrix solution in the dark, and vortexed at 2000 rpm for 10 min to form a homogeneous suspension;

[0007] S3. Spot drying: Use a micropipette to accurately take 1 μL of the mixed solution and vertically drip it onto a polished stainless steel target plate using a 0.5 mm diameter spotting needle. Vacuum dry it in a constant temperature and humidity chamber at -0.1 MPa for 2 hours to form a nanoscale composite crystalline film layer with a thickness of 200-500 nm to obtain the graphene oxide matrix.

[0008] Furthermore, the oxygen content of the graphene oxide powder is 15-20wt%

[0009] Furthermore, the concentration of the graphene oxide dispersion is 0.1-0.5 mg / mL.

[0010] Furthermore, the matrix solution consists of an α-cyano-4-hydroxycinnamic acid solution and an acetonitrile TFA solvent.

[0011] Furthermore, the concentration of the α-cyano-4-hydroxycinnamic acid solution is 10 mg / mL.

[0012] Furthermore, the acetonitrile TFA solvent consists of acetonitrile and 0.1% trifluoroacetic acid aqueous solution in a mass ratio of 7:3.

[0013] Another technical solution adopted in this application is: an optimized matrix for MALDI,

[0014] Furthermore, the optimized matrix of the MALDI is a graphene oxide matrix, and the ratio of carbon atoms to oxygen atoms in the graphene oxide matrix is 2.1:1.

[0015] Furthermore, the oxygen-containing functional groups in the graphene oxide matrix account for 18.7%.

[0016] Furthermore, the absorbance of the graphene oxide matrix at 355 nm in the UV-visible absorption spectrum is 0.85±0.05; the thermal diffusion coefficient of the graphene oxide matrix is 12.7 mm 2 / s.

[0017] The beneficial effects of the present application are as follows: the present application will combine the characteristics of carbon materials and traditional organic matrices, and use graphene oxide with higher uniformity, high hydrophilicity and solid-phase adsorption capacity to assist traditional solid matrices with high ionization ability, so that the analyte and the organic matrix are uniformly adsorbed on the graphene oxide, avoiding the generation of "hot spots", helping the matrix to form more uniform crystals, and improving the reproducibility of MALDI detection. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to specific embodiments.

[0019] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from the description. Therefore, the present invention is not limited to the specific embodiments disclosed in the following specification.

[0020] The present application provides a method for preparing an optimized MALDI matrix, comprising:

[0021] Preparation of graphene oxide dispersion: 50-100 mg of graphene oxide powder was weighed and added to 100 mL of ultrapure water. The mixture was magnetically stirred at 25°C and 800 rpm for 15 minutes. The mixture was then ultrasonicated for 30 minutes to obtain a uniform graphene oxide dispersion.

[0022] S2. Matrix mixing: 1 mL of the graphene oxide dispersion was mixed with 5-10 mL of the auxiliary matrix solution in the dark, and vortexed at 2000 rpm for 10 min to form a homogeneous suspension;

[0023] S3. Spot drying: Use a micropipette to accurately take 1 μL of the mixed solution and vertically drip it onto a polished stainless steel target plate using a 0.5 mm diameter spotting needle. Vacuum dry it in a constant temperature and humidity chamber at -0.1 MPa for 2 hours to form a nanoscale composite crystalline film layer with a thickness of 200-500 nm to obtain the graphene oxide matrix.

[0024] The graphene oxide powder has an oxygen content of 15-20 wt%; the graphene oxide dispersion has a concentration of 0.1-0.5 mg / mL; the matrix solution comprises an α-cyano-4-hydroxycinnamic acid solution and an acetonitrile-TFA solvent. The concentration of the α-cyano-4-hydroxycinnamic acid solution is 10 mg / mL; and the acetonitrile-TFA solvent comprises acetonitrile and a 0.1% trifluoroacetic acid aqueous solution in a mass ratio of 7:3.

[0025] The present application also provides an optimized matrix for MALDI, wherein the optimized matrix for MALDI is a graphene oxide matrix, wherein the ratio of carbon atoms to oxygen atoms in the graphene oxide matrix is 2.1:1. The oxygen-containing functional groups in the graphene oxide matrix account for 18.7% (of which hydroxyl groups account for 11.2%, epoxy groups account for 4.9%, and carboxyl groups account for 2.6%). The absorbance of the graphene oxide matrix at 355nm in the ultraviolet-visible absorption spectrum is 0.85±0.05; the thermal diffusion coefficient of the graphene oxide matrix is 12.7 mm 2 / s.

[0026] The lateral size of the graphene oxide matrix was measured by dynamic light scattering (DLS) to be 72.3 nm ± 15.6 nm (PDI = 0.21), and atomic force microscopy (AFM) showed a monolayer thickness of 1.2 nm ± 0.3 nm; the zeta potential of the graphene oxide matrix was -35 mV ± 2.5 mV (pH 6.8), and the suspension rate after standing for 24 hours was > 98% (conventional carbon materials < 85%).

[0027] Compared with traditional auxiliary matrices, the crystallization data obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI) on graphene oxide matrices using the above method include:

[0028] (1) Laser confocal microscopy showed that the coefficient of variation of crystal density decreased from 0.48 in the traditional matrix to 0.15, a decrease of 68.7% compared with the traditional matrix;

[0029] (2) When time-of-flight mass spectrometry was used to detect enzymatic peptide fragments of bovine serum albumin (BSA), the RSD of the characteristic peak signal intensity at m / z 927.48 decreased from 17.3% to 4.1% (n=20);

[0030] (3) MALDI was performed using the optimized matrix of this application, and the effective crystallization sites per square millimeter reached 1.2×10 4 The effective crystallization sites of traditional matrix are 3.5×10 3 The matrix-assisted efficiency is greatly increased; among them, the laser energy threshold required for the optimized matrix of the present application is reduced to 28μJ±3μJ, while the traditional matrix requires 45μJ±8μJ.

[0031] Table 1 MALDI matrix optimization scheme and performance comparison

[0032]

[0033] Example 1

[0034] The pretreatment process of graphene oxide as a MALDI-assisted matrix is as follows (taking traditional Chinese medicine saponins as an example):

[0035] Preparation of saponin standard solution: Weigh 5.00 mg each of ginsenoside Rb1, ginsenoside Re and ginsenoside R1 to prepare a stock solution equivalent to 1.0 mmol / mL, and store in a refrigerator at 40°C.

[0036] Preparation of internal standard stock solution: Weigh 5 mg of Sanguisorba officinalis saponin I and prepare a stock solution equivalent to 1.0 mmol / mL. Store in a refrigerator at 4°C and dilute to 400 nmol / mL before use.

[0037] Preparation of MALDI matrix solution: Accurately weigh 5 mg of DHB (dihydroxybenzoic acid) and dissolve it in 1 mL of 70% ethanol. Weigh 10 mg of graphene oxide (GO) and dissolve it in 10 mL of ethanol-ultrapure water (7:3, v / v) and sonicate for 10 minutes. After 24 hours of rest, the upper graphene oxide suspension is diluted to 0.2 mg / mL for later use. The DHB-GO combined matrix is prepared by mixing equal volumes of 5 mg / mL DHB solution and 0.2 mg / mL GO suspension.

[0038] MADLI-TOF / MS conditions:

[0039] Before each data acquisition, calibration was performed using standard peptides.

[0040] Ion detection mode: positive ion detection mode

[0041] Detection range: m / z 500-1200

[0042] Tuning mode: Reflection mode

[0043] Ion source voltage: 20 kV; pulse voltage: 2625 V; reflection voltage: 24.4 kV; line mode detector voltage: 2.7 kV; reflection mode detector voltage: 1.98 kV; maximum laser repetition rate: 30 Hz;

[0044] Laser energy: DHB: 65; DHB-GO: 60

[0045] Signal acquisition method: The instrument automatically performs rasterized laser scanning of the target, with 100 shots for each target hole, and the image is composed of the average value of the 100 shots.

[0046] Table 1-1 Sensitivity and precision of ginsenoside Rb1, Re and notoginsenoside R1 determined by MALDI-TOF mass spectrometry using DHB or DHB-go as the matrix.

[0047] Table 2 shows the comparison of sensitivity and precision of ginsenoside Rb1, Re and notoginsenoside R1 by MALDI-TOF mass spectrometry

[0048]

[0049] ***P<0.001

[0050] Results of DHB-GO combined with matrix to improve the reproducibility of saponin:

[0051] As shown in Table 2, to compare the shot-to-shot reproducibility of the DHB-GO matrix with that of DHB, seven points were randomly selected within a single spot to measure the peak intensity variations of Rb1, Re, and R1 within each shot. The coefficients of variation (CV) of the peak intensities of Rb1, Re, and R1 were all above 65% when using DHB as the matrix. In contrast, no significant sweet spot was observed in DHB-GO, and the CV values for the peak intensities of Rb1, Re, and R1 within each shot were all less than 25%.

[0052] To investigate the spot-to-spot reproducibility of the DHB-GO matrix and DHB, the peak intensity variations of Rb1, Re, and R1 were recorded for seven spots in each matrix (Table 2). The coefficients of variation (CV) for the peak intensities of Rb1, Re, and R1 in the DHB matrix were 26.21%, 28.28%, and 25.61%, respectively. In contrast, the CV values for the peak intensities of Rb1, Re, and R1 in each spot of DHB-GO were all less than 14%.

[0053] Both data directly indicate that the DHB-GO combined matrix can form a uniform mixture surface of the matrix and the analyte, making MALDI detection unnecessary to find "hot spots", reducing the signal difference between points, and greatly improving the quantitative repeatability of MALDI-MS.

[0054] The above description is merely an embodiment of the present application and does not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the present application specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for preparing an optimized matrix for MALDI, characterized in that: The following steps are involved: Preparation of graphene oxide dispersion: 50-100 mg of graphene oxide powder was weighed and added to 100 mL of ultrapure water. The mixture was magnetically stirred at 25°C and 800 rpm for 15 minutes. The mixture was then ultrasonicated for 30 minutes to obtain a uniform graphene oxide dispersion. S2. Matrix mixing: 1 mL of the graphene oxide dispersion was mixed with 5-10 mL of the auxiliary matrix solution in the dark, and vortexed at 2000 rpm for 10 min to form a homogeneous suspension; S3. Spot drying: Use a micropipette to accurately take 1 μL of the mixed solution and vertically drip it onto a polished stainless steel target plate using a 0.5 mm diameter spotting needle. Vacuum dry it in a constant temperature and humidity chamber at -0.1 MPa for 2 hours to form a nanoscale composite crystalline film layer with a thickness of 200-500 nm to obtain the graphene oxide matrix.

2. The method according to claim 1, characterized in that The oxygen content of the graphene oxide powder is 15-20 wt%.

3. The method according to claim 1, characterized in that The concentration of the graphene oxide dispersion is 0.1-0.5 mg / mL.

4. The method according to claim 1, wherein The matrix solution consists of α-cyano-4-hydroxycinnamic acid solution and acetonitrile TFA solvent.

5. The method according to claim 4, characterized in that The concentration of the α-cyano-4-hydroxycinnamic acid solution is 10 mg / mL.

6. The method according to claim 4, characterized in that The acetonitrile TFA solvent consists of acetonitrile and a 0.1% trifluoroacetic acid aqueous solution in a mass ratio of 7:

3.

7. An optimized matrix for MALDI, characterized in that The optimized matrix of the MALDI is a graphene oxide matrix, and the ratio of carbon atoms to oxygen atoms in the graphene oxide matrix is 2.1:

1.

8. The optimized MALDI matrix according to claim 7, characterized in that: The oxygen-containing functional groups in the graphene oxide matrix account for 18.7%.

9. The optimized MALDI matrix according to claim 7, characterized in that: The absorbance of the graphene oxide matrix at 355 nm in the UV-visible absorption spectrum is 0.85±0.05; the thermal diffusion coefficient of the graphene oxide matrix is 12.7 mm 2 / s.

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