Chromatography-mass spectrometry analysis method for simultaneously detecting 48 RNA modifications

Abstract

The invention discloses a chromatography-mass spectrometry analysis method for simultaneously detecting 48 RNA modifications. By optimizing chromatographic separation conditions and mass spectrum parameters, identification and analysis of eight groups of isomers and eight groups of RNA modifications with similar molecular weight transformation are realized. And establishing a standard curve by taking the ratio of the peak area of the RNA modified standard solution to the internal standard peak area as a vertical coordinate and the amount of the RNA modified substance as a horizontal coordinate, and quantifying the content of the RNA modification in the sample solution by using an internal standard method. The method disclosed by the invention is simple, convenient, rapid, accurate, good in repeatability and reliable in result, and can be used for detecting and analyzing RNA modification in a biological sample.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis and detection, and specifically relates to a chromatography-mass spectrometry analysis method for simultaneously detecting 48 kinds of RNA modifications. Background Art

[0002] In addition to the four standard nucleosides adenosine (A), guanosine (G), cytidine (C) and uridine (U), researchers discovered the first RNA modification in yeast as early as 1957. So far, more than 170 chemical modifications have been identified in various types of RNA in different organisms. Many RNA modifications are dynamically reversible and play a key role in regulating RNA generation, stability, folding, transport and metabolism.

[0003] To date, a variety of methods have been used for the qualitative and quantitative analysis of RNA modifications, such as two-dimensional thin layer chromatography, dot blot, and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Compared with other methods, LC-MS / MS has become a powerful tool for identifying and quantifying RNA modifications due to its high sensitivity, high selectivity, and high accuracy. However, most current LC-MS / MS methods are limited to the analysis of a few RNA modifications, and there is no method that can achieve the simultaneous detection and analysis of dozens of RNA modifications.

[0004] Currently, there is a lack of a chromatography-mass spectrometry method that can simultaneously separate and quantify dozens of RNA modifications. Summary of the invention

[0005] In view of the above technical problems, the object of the present invention is to provide a chromatography-mass spectrometry analysis method for simultaneously detecting 48 types of RNA modifications.

[0006] The present invention achieves separation and identification of 48 kinds of RNA modifications by optimizing chromatographic separation conditions and mass spectrometry parameters, including identification and analysis of 8 groups of isomers and 8 groups of RNA modifications with similar molecular weight transitions. A standard curve is established with the ratio of the peak area of ​​the RNA modification standard solution to the internal standard peak area as the ordinate and the amount of the RNA modified substance as the abscissa, and the content of the RNA modification in the sample solution is quantified by the internal standard method.

[0007] The technical solution provided by the present invention is as follows:

[0008] A chromatography-mass spectrometry method for simultaneous detection of 48 RNA modifications

[0009] The 48 RNA modifications include:

[0010] 8 groups of isomers: m1 A and m 6 A;io 6 A(cis) and io 6 A(trans);m 1 G、m 2 G and m 7 G;m 3 C and m 5 C; f 5 C and m 4,4 C;s 2 U.s 4 U and ho 5 U;m 3 U and m 5 U;mo 5 U and m 5 s 2 U;

[0011] 8 groups of RNA modifications with similar molecular weight transitions: m 1 A.m 6 A and m 1 I; Am and Im; Cm and Um; m 3 C.m 5 C.m 3 U and m 5 U;m 3 U, m 5 U and s 2 C;s 2 C.ho 5 U.s 2 U and s 4 U;hm 5 C. mo 5 U and m 5 s 2 U;m 5 Cm and m 5 Um;

[0012] The analysis steps are as follows:

[0013] Step (1): using liquid chromatography-tandem mass spectrometry to perform gradient separation by optimizing chromatographic conditions and mass spectrometry parameters;

[0014] The HPLC parameters are as follows:

[0015] Chromatographic column: Shimadzu Shim-pack GIST C18 column;

[0016] Mobile phase: Phase A: NH4HCO3; Phase B: methanol;

[0017] Sample preparation solvent: pure water;

[0018] The mass spectrometry parameters were as follows: a) ion source: electrospray ionization; b) scanning mode: multiple reaction monitoring mode; c) DL temperature: 250 °C;

[0019] Step (2): Prepare a standard solution, establish a standard curve, use the internal standard method to quantify the absolute content of RNA modification in the sample solution, and calculate its molar ratio.

[0020] Furthermore, the chromatographic column has a length of 100 mm, a diameter of 2.1 mm, and a particle size of 2.0 μm.

[0021] Furthermore, the concentration of phase A is 2-5 mM. Preferably, the concentration of phase A is 2 mM.

[0022] Furthermore, the operating temperature of the chromatographic column is 40° C., and the flow rate is 0.2-0.3 mL / min. Preferably, the operating temperature of the chromatographic column is 40° C., and the flow rate is 0.3 mL / min.

[0023] Furthermore, the injection volume was 20 μL.

[0024] Further, the gradient separation procedure of liquid chromatography is:

[0025]

[0026] Furthermore, the gradient separation procedure of liquid chromatography is:

[0027]

[0028] Further, the step (2) comprises the following steps: preparing a series of nucleosides containing different amounts of substances and a certain amount of isotope internal standard rC- 13 C5 mixed standard; with the ratio of the peak area of ​​the nucleoside standard solution to the internal standard peak area as the ordinate, and the amount of nucleoside substance as the abscissa to establish a standard curve, the absolute content of RNA modification in the sample solution is quantified by the internal standard method, and it is normalized to the corresponding normal nucleoside to obtain the molar ratio (%).

[0029] The beneficial effects of the present invention are as follows:

[0030] 1) The method provided by the present invention is simple to operate, fast and efficient, does not require cumbersome processing steps, and the entire experimental process can be completed within one day, which is conducive to promotion and use.

[0031] 2) The present invention can realize the identification and analysis of 8 groups of isomers and 8 groups of RNA modifications with similar molecular weight transitions.

[0032] 3) The present invention can perform quantitative analysis of global RNA modification levels very accurately.

[0033] 4) The present invention can be directly used for the analysis of RNA modification levels in clinical samples, providing a powerful tool for in-depth research on the functional studies and synergistic effects of RNA modifications. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The following are the structural formulas of 48 RNA modification products in the present invention.

[0035] Figure 2 The extracted ion chromatograms of 48 RNA modification standards in the present invention.

[0036] Figure 3 In the present invention, 10 RNA modifications (m 1 A.m 6 A.m 6 Am, Am, m 7 G, Gm, m 3 C.m 5 (A, D, G) Extracted ion chromatograms of 10 modified nucleosides in mRNA of thyroid cancer patients. (B, E, H) Extracted ion chromatograms of 10 modified nucleoside standards. (C, F, I) Enzyme blank.

[0037] Figure 4 The contents of 10 RNA modifications in mRNA of thyroid cancer and corresponding adjacent tissues. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific implementation examples, but they should not be construed as limitations on the present invention. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0039] Unless otherwise specified, the technical means used in the examples, including extraction of mRNA from tissue samples and enzymatic hydrolysis of RNA, are conventional means well known to those skilled in the field of biotechnology.

[0040] Example 1

[0041] The steps for optimizing the chromatographic mass spectrometry conditions of 48 RNA modifications are as follows: First, optimize the chromatographic separation conditions of the 48 modifications. Prepare a mixed solution of standards containing 48 RNA modifications, and optimize the chromatographic conditions of nucleosides under the conditions of 2mM NH4HCO3 (phase A) and methanol (phase B) as the mobile phase. Then, divide the 48 modifications into five groups to prepare mixed standard solutions, and optimize the multiple reaction monitoring (MRM) parameters of each modified nucleoside. The final optimized MRM parameters are shown in Table 1.

[0042] The HPLC parameters are as follows:

[0043] Chromatographic column: Shimadzu Shim-pack GIST C18 column (100 mm × 2.1 mm i.d., 2.0 μm);

[0044] Mobile phase: Phase A: 2mM NH4HCO3; Phase B: methanol;

[0045] Sample preparation solvent: pure water

[0046] Column temperature: 40°C;

[0047] Flow rate: 0.3mL / min.

[0048] Gradient separation procedure:

[0049]

[0050] In gradient separation procedures, all ratios are by volume.

[0051] Mass spectrometry parameters:

[0052] The mass spectrometry parameters are as follows: a) ion source: electrospray ionization; b) scanning mode: multiple reaction monitoring mode; c) DL temperature: 250°C. Ten mixed standard solutions were prepared, each containing about 5 nucleoside standards, and the mixed standard solutions were automatically optimized by the instrument.

[0053] The optimized chromatographic separation conditions and mass spectrometry parameters enabled the separation and identification of 48 RNA modifications, including 8 groups of isomers (m 1 A and m 6 A;io 6 A(cis) and io 6 A(trans);m 1 G、m 2 G and m 7 G;m 3 C and m 5 C; f 5 C and m 4,4 C;s 2 U.s 4 U and ho 5 U;m 3 U and m 5 U;mo 5 U and m 5 s 2 U) and 8 groups of RNA modifications with similar molecular weight transitions (m 1 A.m 6 A and m 1 I; Am and Im; Cm and Um; m 3 C.m5 C.m 3 U and m 5 U;m 3 U, m 5 U and s 2 C;s 2 C.ho 5 U.s 2 U and s 4 U;hm 5 C. mo 5 U and m 5 s 2 U;m 5 Cm and m 5 Identification and analysis of Um Figure 2 ).

[0054] Table 1 MRM parameters for LC-MS / MS analysis of nucleosides

[0055]

[0056]

[0057] Example 2

[0058] Detection and quantitative analysis of RNA modifications in mRNA of cancer tissues and adjacent tissues of thyroid cancer patients

[0059] The specific steps are as follows:

[0060] (1) Detection of RNA modifications in mRNA of cancer tissues and adjacent tissues of thyroid cancer patients. Twenty-six thyroid cancer tissues and corresponding adjacent tissues from 13 thyroid cancer patients from Hubei Cancer Hospital (Wuhan, China) were collected and mRNA was isolated from them. The isolated mRNA was hydrolyzed into nucleosides and subsequently analyzed by LC-MS / MS under the above-mentioned chromatography-mass spectrometry conditions. The results showed that 10 RNA modifications were detected in mRNA: m 1 A.m 6 A.m 6 Am, Am, m 7 G, Gm, m 3 C.m 5 C, Cm and I ( Figure 3 ).

[0061] (2) Establishment of standard curve. In order to quantitatively analyze RNA modification in thyroid cancer tissue mRNA, a series of RNA modification samples containing different amounts of substances and a certain amount of isotope internal standard rC- 13C5 mixed standard. After the mixed standard was analyzed by mass spectrometry, a series of standard curves were drawn with the obtained peak area ratio (RNA modification / isotope internal standard) as the ordinate and the amount of RNA modified substance as the abscissa. The standard curve drawing method for normal nucleosides is the same as above. As shown in Table 2, the linear correlation coefficients of the standard curves of all nucleosides are R 2 All of them are greater than 0.99, indicating that the method has good linearity.

[0062] Table 2 Standard curve for LC-MS / MS analysis of RNA modifications

[0063]

[0064] (3) Methodological validation

[0065] 1) Intraday and interday precision

[0066] In the linear range of each standard curve, three concentration points, low, medium and high, were selected, and the injection was repeated three times for three consecutive days under the above chromatographic conditions to measure the intra-day and inter-day precision. The results showed that the intra-day and inter-day relative standard deviation (RSD) was less than 12.7%, which met the basic requirements of the analysis.

[0067] 2) Limit of detection and limit of quantification

[0068] The amount corresponding to a signal-to-noise ratio of 3 was defined as the limits of detection (LODs), and the amount corresponding to a signal-to-noise ratio of 10 was defined as the limits of quantification (LOQs). The LODs of RNA-modified nucleosides used in this experiment ranged from 0.20 to 5.48 fmol, and the LOQs ranged from 0.66 to 18.25 fmol.

[0069] 3) Quantitative analysis of RNA modification in mRNA of cancer tissues and adjacent tissues of thyroid cancer patients

[0070] The validated LC-MS / MS method was used to quantitatively analyze 10 RNA modifications detected in mRNA of 26 thyroid cancer and corresponding adjacent tissues from 13 thyroid cancer patients. Figure 4 ) showed that compared with the corresponding adjacent tissues, the mRNA in thyroid cancer tissues was 5 The content of C increased significantly.

[0071] The above description is only a preferred specific implementation manner of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the scope of protection of the invention.

Claims

1. A chromatography-mass spectrometry method for simultaneously detecting 48 RNA modifications, characterized in that: The 48 RNA modifications include: 8 groups of isomers: m 1 A and m 6 A;io 6 A(cis) and io 6 A(trans);m 1 G、m 2 G and m 7 G;m 3 C and m 5 C; f 5 C and m 4, 4 C;s 2 U.s. 4 U and ho 5 U;m 3 U and m 5 U;mo 5 U and m 5 s 2 U; 8 groups of RNA modifications with similar molecular weight transitions: m 1 A.m 6 A and m 1 I; Am and Im; Cm and Um; m 3 C.m 5 C.m 3 U and m 5 U;m 3 U, m 5 U and s 2 C;s 2 C.ho 5 U.s. 2 U and s 4 U;hm 5 C. mo 5 U and m 5 s 2 U;m 5 Cm and m 5 Um; The analysis steps are as follows: Step (1): using liquid chromatography-tandem mass spectrometry to perform gradient separation by optimizing chromatographic conditions and mass spectrometry parameters; The HPLC parameters are as follows: Chromatographic column: Shimadzu Shim-pack GIST C18 column; Mobile phase: Phase A: NH4HCO3; Phase B: methanol; Column oven temperature: 40°C Sample preparation solvent: pure water; The mass spectrometry parameters were as follows: a) ion source: electrospray ionization; b) scanning mode: multiple reaction monitoring mode; c) DL temperature: 250 °C; Step (2): Prepare a standard solution, establish a standard curve, use the internal standard method to quantify the absolute content of RNA modification in the sample solution, and calculate its molar ratio.

2. The chromatography-mass spectrometry analysis method for simultaneously detecting 48 RNA modifications according to claim 1, characterized in that: The chromatographic column has a length of 100 mm, a diameter of 2.1 mm, and a particle size of 2.0 μm.

3. The chromatography-mass spectrometry analysis method for simultaneously detecting 48 RNA modifications according to claim 1, characterized in that: The concentration of the A phase is 2-5 mM.

4. The chromatography-mass spectrometry analysis method for simultaneously detecting 48 RNA modifications according to claim 1, characterized in that: The operating temperature of the chromatographic column is 40° C., and the flow rate is 0.2-0.3 mL / min.

5. The chromatography-mass spectrometry analysis method for simultaneously detecting 48 RNA modifications according to claim 1, characterized in that: The injection volume was 20 μL.

6. The chromatography-mass spectrometry analysis method for simultaneously detecting 48 RNA modifications according to claim 1, characterized in that: The gradient separation procedure for liquid chromatography is:

7. The chromatography-mass spectrometry analysis method for simultaneously detecting 48 RNA modifications according to claim 6, characterized in that: The gradient separation procedure for liquid chromatography is:

8. The chromatography-mass spectrometry analysis method for simultaneously detecting 48 RNA modifications according to claim 1, characterized in that: The step (2) comprises the following steps: preparing a series of nucleosides containing different amounts of substances and a certain amount of isotope internal standard rC- 13 C5 mixed standard; with the ratio of the peak area of ​​the nucleoside standard solution to the internal standard peak area as the ordinate and the amount of nucleoside substance as the abscissa to establish a standard curve, the absolute content of RNA modification in the sample solution is quantified by the internal standard method, and it is normalized to the corresponding normal nucleoside to obtain the molar ratio.

Images