As an analytical method for mass spectrometry detection, multiple reaction monitoring (MRM) technology has the outstanding advantages of strong specificity, high sensitivity, high accuracy, good reproducibility, wide linear dynamic range, and automated high-throughput. , these qualities can meet the urgent needs of many research fields today. Real-time quantitative monitoring through MRM technology can be used for pharmacokinetic research, clinical diagnosis, illegal drug inspection, industrial quality control such as food and cosmetics, environmental testing, agricultural botany research, and discovery of metabolomics and proteomics Biomarker. Research.
1. The principle of mass spectrometry MRM technology
MRM technology is a method based on known or assumed reactive ion information, targeted selection of data for mass spectrometry signal acquisition, signal recording of ions that conform to the rules, removal of the interference of ions that do not conform to the rules, and statistical analysis of the data. Mass spectrometry technology to obtain quantitative information of mass spectrometry. MRM technology is evolved on the basis of single reaction monitoring (SRM) technology. For MRM technology, the key is to firstly be able to detect specific precursor ions, then only the selected specific precursor ions are collision-induced, and finally the interference of other product ions is removed, and only the selected specific precursor ions are collision-induced. The specific product ions were collected for mass spectral signal acquisition. Since triple quadrupole system (TQS) is the most sensitive mass spectrometer system for single mass-to-charge ratio scanning, it is the most suitable mass spectrometer for MRM analysis.
2. Characteristics of Mass Spectrometry MRM Technology
Features of MRM technology: ①High sensitivity: Through two-stage ion selection, a large number of interfering ions are eliminated, the chemical background of the mass spectrometer is reduced, and the signal-to-noise ratio of the target detection substance is significantly improved, thereby achieving high detection sensitivity. ②Good reproducibility: In the selective acquisition of mass spectrometry signals by MRM technology, the ionization of the molecule to be tested, the suppression of mass spectrometry signals and the influence of the collision fragmentation process in the source are avoided, so the reproducibility is also improved accordingly. ③High accuracy: Using the specificity of MRM technology, continuous enhanced ion scanning analysis is performed to obtain high-resolution tandem mass spectrometry (MS/MS) fragmentation data, which reduces the analysis process compared with full scan and neutral loss MS scanning modes. The false positive rate of qualitative results ensures the accuracy of the analysis. ④High throughput: Using the most advanced mass spectrometry system, MRM technology can process up to 300 parent ion-daughter ion pairs per working cycle. Opportunities to better meet the research needs of proteomics.
3. Application of Mass Spectrometry MRM Technology in Quantitative Analysis
3.1 Application of MRM technology in quantitative analysis of small chemical molecules
MRM quantitative analysis technology has been used in chemical small molecule analysis for more than 30 years. It was first reported in 1978 for isotopic studies of chlorine, and a year later this technology was applied to metabolic detection of blood drug concentrations. Since the advent of triple quadrupole mass spectrometry, MRM technology has become an important method for the analysis of low molecular weight chemicals. Especially in complex drug metabolism research, MRM has become a key core technology. MRM can perform real-time monitoring of prodrugs and their metabolites with high accuracy and high sensitivity. Since then, it has been applied in the analysis of a large number of biologically relevant small molecule chemicals. For example: tracking of endogenous substances, monitoring of therapeutic agents and their metabolites, inspection of illegal drugs and detection of environmental toxicants, etc. With the deciphering of the genome, metabolomics and proteomics came into being, and the research field of life sciences has been greatly expanded since then.
3.2 Application of MRM technology in quantitative analysis of metabolomics
Metabolomics is the science of the holistic and comprehensive analysis of metabolites in biochemical processes. Although the application of MRM technology extends from small molecules to the analysis of endogenous and exogenous metabolites, it is still limited by the metabolomics research itself. The metabolome is complex and varied. It is estimated that there are more than 7,000 molecules in the human body, so the wide area of the metabolome is beyond the ability of mass spectrometry detection. However, the emergence of predictive metabolism and selective metabolomes has drawn researchers’ attention to the concept of targeted scanning, which enables real-time scanning monitoring of a large number of metabolites by applying a variety of selected ion monitoring techniques in MRM mode. Although MRM technology cannot be used as the main scanning method of metabolomics, the emergence of QTRAP mass spectrometry system has provided MRM technology with a broader idea for the research of targeted proteomics discovery stage.
3.3 Application of MRM technology in quantitative analysis of proteomics
The research object of proteomics is a whole that changes dynamically in time and space with extreme complexity. With the deepening and development of proteomics research, especially the progress of differential proteomics research, a large number of functional proteins and potential disease protein markers have been discovered and identified. How to further probe the expression abundance of these proteins to clarify Its function and significance in disease research have become increasingly important. Only relying on the technical route of large-scale protein separation and identification (two-dimensional gel electrophoresis technology to separate proteins, mass spectrometry technology to identify proteins) can no longer meet the needs of these studies, so there is an urgent need for research methods with higher sensitivity and higher selectivity. As a highly specific and sensitive mass spectrometry data acquisition method, MRM technology can play an important role in more targeted research in proteomics, and has gradually attracted the attention of more researchers.
The interference of co-elution of complex components and the wide dynamic range of more than 9 orders of magnitude have always been the key factors affecting the accurate quantification of proteins and peptides in biological fluids such as serum or plasma. Although researchers have also tried to adopt a variety of methods, including chromatographic separation as much as possible, removal of high-abundance proteins, etc., the sensitivity and precision of mass spectrometry for detection of low-abundance proteins still cannot meet the requirements of analysis. MRM technology has demonstrated the advantages of its method in quantitative proteomics applications. First, the dynamic range of the assay is improved to a certain extent. On the one hand, the selective detection of ions reduces the background signal of complex components and enhances the detection sensitivity of some low-abundance proteins. The selection of high-abundance and low-abundance protein MRM transitions balances the difference in response signals between the two. For example, for high-abundance proteins, select a precursor-product ion pair with a lower response abundance; for a low-abundance protein, select a precursor-product ion pair with a higher degree of response, so as to balance the high and low abundance signals difference. Second, interference from co-elution of complex components is reduced, enhancing the specificity of detection. In addition, the high-throughput characteristics of MRM technology also provide conditions for the simultaneous quantification of multiple proteins. By combining the MRM technology with the isotope dilution method or mTRAQ technology, the known amount of the added internal standard peptide and the real peptide in the sample are respectively labeled, and different parent-daughter ion pairs are selected to obtain different chromatographic detection signals. signal, resulting in absolute quantitative results.
The MRM method has good development prospects, but it also faces huge technical challenges. From the literature reports, we can see that a series of quantitative analysis methods established based on MRM technology have many types, strong variability and wide application range, and different sample pretreatment experimental schemes can be selected according to different research needs. Applicable, so as to give full play to the powerful quantitative analysis potential of MRM technology.