Accurate molecular mass determination of DNA & RNA oligonucleotides

Nie Aiying

Thermo Fisher Scientific (China) Co., Ltd.

Key words

LTQ-Orbitrap Elite; nucleic acid drug; molecular mass determination

1 Introduction

With the deep understanding of nucleic acid structure and function, nucleic acid drugs that specifically bind or cleave disease-causing genes have gradually become a new hotspot in drug research. Nucleic acid drugs have high efficiency and wide application range, and can supplement traditional drugs. And can also play an important role in the early clinical diagnosis. Nucleic acid drugs mainly refer to various oligoribonucleotides (RNA) or oligodeoxyribonucleotides (DNA) with different functions, which mainly act at the gene level and function in the upstream stage of information flow transmission, and the efficiency is relatively high. And by binding or cleavage of specific pathogenic genes, there is no effect on other genes. In the process of information flow, the signal is amplified step by step. One gene can transcribe multiple mRNAs, and one mRNA can translate multiple proteins. Therefore, compared with the final product-protein acting on the information stream, nucleic acid drugs have a wide range. Application prospects. Current nucleic acid drugs can bind to some transcription factors, thrombin, human immunodeficiency virus, special parts of double-stranded DNA, etc., thereby inhibiting DNA replication or binding of proteins, receptors, etc., thereby interfering with and preventing viruses or erroneous genes and Protein expression and amplification, so in the development and production of genetic drugs, the establishment and development of a more efficient, sensitive, rapid and accurate oligonucleotide structure and sequence analysis methods, has always been a biochemist, One of the research topics of medical workers and instrumental analysis companies.

Biomass technology provides a new approach to molecular mass and sequence analysis of oligonucleotides. Previously, matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF) was mainly used for the analysis of oligonucleotides with small molecular mass. The sensitivity, resolution, and alkali metal ion addition peaks were affected by the sensitivity of the instrument. There are certain problems with reliability. In addition, based on the detection of conventional electrospray ion trap (Ion Trap) mass spectrometry, it is also limited by resolution and mass accuracy, and can not meet the detection and analysis of high-precision, high-resolution and highly sensitive oligonucleotides.

The LTQ-Orbitrap Elite combined mass spectrometer combines the latest dual pressure linear ion trap mass spectrometer with the new high field OrbitrapTM mass analyzer to deliver up to 240,000 resolution (FWHM), high sensitivity, fast scanning speed and greater Dynamic range and low-resolution and high-resolution scanning can be achieved simultaneously on the same mass spectrum to meet different customer needs. The system provides accurate, fast, and reliable analytical testing for molecular mass determination of oligonucleotides.

2. Experimental part

2.1 Instruments and reagents

Mass Spectrometry Instruments: LTQ-Orbitrap Elite (Thermo Fisher Scientific, USA);

Chromatography instrument: Accela LC system (Thermo Fisher Scientific, USA);

Column: BioBasic column (C8, 2.1 × 50 mm, 5 μm, 300 Å) (Thermo Fisher Scientific, USA);

Reagents: ammonium acetate, secondary deionized water, chromatographic grade methanol.

2.2 Instrument method

Chromatographic conditions: see Table 1 for details;

Mass spectrometry conditions: see Table 2 for details;

Table 1. Chromatographic conditions for determination of molecular weight of oligonucleotides

Table 2 Mass spectrometric conditions for molecular mass determination of oligonucleotides

2.3 Data analysis methods

The original mass spectrum was deconvoluted using ProMass software to obtain the molecular quality information of the intact oligonucleotide.

3. Results and discussion

3.1 Low-resolution LTQ mass detector for determining the molecular mass of oligonucleotides

3.1.1 Original chromatogram

Using a 5 min gradient analysis, the ion traps collected data and the oligonucleic acids peaked at 3.53 min.

3.1.2 Original mass spectrum

The average spectrum of the oligonucleotide spectrum of the 3.53 min accessory in the above figure is averaged, and the average mass spectrum of the oligonucleotide is obtained. Since the resolution of the LTQ is limited, the charge cannot be directly distinguished, so it needs to be performed by software. Deconvolution treatment to obtain the molecular mass of the oligonucleotide.

3.1.3 Mass spectrometry results after deconvolution

The average molecular mass after deconvolution by ProMass software was 9247.3, which is exactly the same as the theoretical average molecular mass, and the ProMass software assigned the charge of the mass spectrum peak in the original mass spectrum, as shown below, with a mass-to-charge ratio of 1540.2. The peak corresponds to a charge of –6 and the mass spectral peak with a mass-to-charge ratio of 1848.5 corresponds to a charge of –5.

3.2 High-resolution Orbitrap mass analyzer for determining the molecular mass of oligonucleotides

3.2.1 Original chromatogram

Compared with the low-resolution experiment, only by changing the resolution of mass spectrometry, the chromatographic retention time of the oligonucleotide can be seen unchanged, basically at 3.52 min. Peak, but as the resolution of the mass spectrometer increases, the original mass spectrum changes significantly. As shown below, when the linear ion trap LTQ is used as the mass detector, only one large peak is detected and the charge cannot be resolved. When Orbitrap is used as the quality detector, when the resolution R is set to 15000, only one large peak is detected, and the charge is still indistinguishable. When Orbitrap is used as the quality detector, the resolution R is set to 30,000, which can be observed. Some isotope peaks, the charge can be resolved; when using Orbitrap as the mass detector, the resolution R is set to 60,000, a slightly better isotope peak can be observed, but not completely separated, the charge can also be resolved; when using Orbitrap as Mass detector, when the resolution R is set to 120,000, the isotope peak can be completely separated, the charge is the same When using Orbitrap as the mass detector, the resolution R is set to 240,000, the isotope peak can be completely separated, and the peak shape is sharper, and the charge can also be resolved, so we can accurately calculate The monoisotopic mass and average mass of the oligonucleotide, the final experimental results are completely consistent with the theoretically predicted molecular mass.

Similarly, we performed mass spectrometry tests on several different molecular mass oligonucleotides with the following results:

From the above mass spectrum, it can be seen that as the resolution of the mass spectrum increases, the isotope peaks of the oligonucleotides can be completely separated, and the peak width is narrower, the charge can be completely resolved, and software-assisted, The monoisotopic mass and average molecular mass of the oligonucleotide can be directly calculated, making the analytical test of the oligonucleotide faster and more direct.

4 Conclusion

In this paper, an online method for molecular mass spectrometry of oligonucleic acid drugs was established by LTQ-Orbitrap Elite mass spectrometry, which can be used to determine oligonucleotides more efficiently, sensitively, rapidly and accurately in the development and analysis of nucleic acid drugs and real-time production detection. Molecular mass. The experimental results show that the LTQ-Orbitrap Elite mass spectrometer greatly improves and promotes gene drugs with its ultra-high resolution, ultra-fast scanning speed, high mass accuracy, ultra-low sensitivity and large dynamic range. Identification analysis.