TALENT EDUCATION

The Tsinghua team realized the analysis of high-resolution biomolecular isomers


Tsinghua News Network, March 21 — The correlation between the structural analysis of biomolecules and related biological functions has become the frontier of life sciences. Biomolecules have a multi-level structure, and an important factor in their structural complexity is molecular heterogeneity. Different isomeric molecules (Isomers and isoforms) have the same chemical formula and molecular weight, but different chemical structures. For example, monosaccharides have a variety of isomers, including glucose, fructose, galactose, etc.; Polysaccharides are composed of two pairs of monosaccharides connected to each other by glycosidic bonds, resulting in more complex structural isomerism (different order of atoms or groups of atoms in molecules, structural or constitutional isomers) and stereoisomerism (the same order of connection but different spatial rankings, spatial isomers or stereoisomers).
Ion mobility (IM) and mass spectrometry (MS) combined (IM-MS) analysis has been developed as a major means for the structural elucidation of biomolecules, especially biological macromolecules, and has become the main direction of the development of mass spectrometry instruments. IM can distinguish isomers or isobars that cannot be distinguished by MS, which is a unique property that is very important for the study of the structure of biomolecules, and has been widely used in the study of sugar structure, lipid structure, protein structure and activity, and protein-molecule interaction in recent years. In recent years, a variety of IM analysis methods have been proposed, such as Drift time ion mobility spectrometry, trapped ion mobility spectrometry, and traveling wave TWIMS spectrometry) and asymmetric field FAIMS (Field asymmetric ion mobility spectrometry). However, these technologies are based on the principle of low E/N field (E/N < 30 Td, E represents the electric field strength, N represents the neutral gas number density, Td is the Townsend number), and the separation resolution is generally 40-200, which is not enough to solve the urgent needs of biomolecular isomer analysis research.
In view of the above problems, the biomedical instrument and application research team of the Department of Precision Instrumentation of Tsinghua University sought to break through the limitations of low resolution of ion mobility analysis in the high E/N field, proposed an ultra-high field ion cloud scanning technology, and realized high-resolution IM analysis with a migration resolution of more than 10,000 on the Mini β mass spectrometry instrument system (PURSPEC Technology (Beijing) Co., Ltd.), which is more than an order of magnitude higher than the existing technology (Figure 1). Ultra-high field ion cloud scanning technology adopts the physical principle of forced oscillation to realize the ion cloud separation of isomer ions under ultra-high field (about 1×105Td) conditions, and high-resolution IM spectra of isomer ions can be obtained by scanning the excitation oscillation voltage.
20230315-Instrument setting, principle and performance characterization of ion cloud structure analysis technology-Wang Zhuofan-Biomedical Instrument and Application Team, Department of Precision Instrumentation, Tsinghua University, reported .jpg
Figure 1.Instrument setup, principle, and performance characterization of ion cloud scanning analysis technology. (a) Mini β mass spectrometry instrument system. (b) Schematic diagram of the experimental setup. (c) Principle of ion cloud scanning technology. Ion trajectories of two isomer ions (violet and blue) under forced vibration. (d) Ion cloud scan spectra obtained
Four disaccharide isomers (trehalose, maltose, cellodisaccharide, and lactose, Figure 2A) were structurally analyzed using high-field ion cloud scanning analysis (Figure 2B), and the mixture of lactose and cellobiose was scanned (Figure 2C) and compared with traditional tandem mass spectrometry (Figure 2D). As can be seen from Figure 2d, lactose and cellobiose have the same fragmentation pattern and cannot be distinguished by tandem mass spectrometry. However, the two isomers can be completely separated by ion cloud scanning (Figure 2C). In addition, the ion cloud scanning analysis technique also exhibited excellent quantitative analysis characteristics (Figures 2e and 2f).
20230315-Disaccharide isomer analysis-Wang Zhuofan-Biomedical instrument and application team of Tsinghua University's precision instrument department reported .jpg
Figure 2. Disaccharide isomer analysis. (a) Four disaccharide isomers and their (b) ion cloud scanning spectra. (C) ion cloud scanning spectra and (d) tandem mass spectrometry spectra of lactose and cellodisaccharide mixtures. (e) Quantitative analysis of two disaccharide standards and (f) mixtures
20230315-Analysis of lipid and peptide isomers-Wang Zhuofan-Biomedical instrument and application team, Department of Precision Instrumentation, Tsinghua University, reported .jpg
Figure 3.Analysis of lipid and peptide isomers. (a) Schematic diagram of lipid isomerism. Ion cloud scanning spectra of various lipid isomers: (b) SN isomer, (c) carbon-carbon double bond position isomerism, and (d) double bond cis- and trans- isomerism. (e) Schematic diagram of the different types of post-translational modifications of peptides and their isomeric patterns. Ion cloud scanning spectra of peptide isoforms for different post-translational modification types: (f) methylation, (g) acetylation, and (h) phosphorylation
Ion cloud scanning technology has universal applicability to various biomolecular isomers. As shown in Figure 3, this technique also resolves lipid and peptide molecular isomers. In the research work, the ion cloud scanning method shows a variety of advantages, such as simple structure of the analysis parts, convenient operation, and powerful temporal/spatial cascade mass spectrometry capabilities, etc., which can be easily combined with multiple types of mass analyzers for the design of hybrid tandem analysis mass spectrometry instruments, which shows a good application prospect in the analysis of complex structures of biomolecules.
The research results were recently published in Nature Communications under the title “High-Resolution Separation of Bioisomers Using Ion Cloud Profiling”.
The first author of the paper is Associate Professor Zhou Xiaoyu of the Department of Precision Instrumentation of Tsinghua University, the corresponding author is Professor Ouyang Zheng, other authors include Wang Zhuofan and Fan Jingjin, 2020 doctoral students of the Department of Precision Instrumentation, and the first completion unit is the State Key Laboratory of Precision Testing Technology and Instrumentation, Department of Precision Instruments, Tsinghua University. The research was greatly assisted by Professor Fang Yu from the Department of Chemistry, Associate Professor Ma Xiaoxiao from the Department of Precision Instrumentation, and Assistant Professor Zhang Wenpeng from the Department of Chemistry of Tsinghua University. The research was supported by the National Natural Science Foundation of China and the Precision Medicine Research Program of Tsinghua University.
Paper Link:
https://www.nature.com/articles/s41467-023-37281-7

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