In the research paradigm of traditional metabolomics (including lipidomics, the same below), non targeted metabolomics aims to use high-resolution mass spectrometry to obtain the complete metabolic profile of samples, providing a broad view of small molecule metabolites; Targeted metabolomics mainly uses triple quadrupole mass spectrometry to achieve precise quantification of metabolites of interest. With the increasing demand for large-scale sample analysis, researchers have begun to seek a balance between high coverage of untargeted metabolomics and high precision of targeted analysis, pioneering large-scale metabolomics techniques (including broad targeted and quasi targeted metabolomics, which will not be distinguished in this article).

Large scale metabolomics focuses on more than a thousand core metabolites in organisms, and directly collects samples using selective reaction monitoring (SRM) or multiple reaction monitoring (MRM) modes on the triple quadrupole mass spectrometry platform. While ensuring metabolite coverage, it can significantly improve the detection sensitivity and specificity of low abundance metabolites, bid farewell to the problem of massive information processing in non targeted analysis, provide a wider dynamic range, more robust data reproducibility, and relative quantitative accuracy for metabolite detection, and provide a solid and reliable data foundation for large-scale sample analysis. However, limited by the scanning speed of triple quadrupole mass spectrometry, typically only 1-2 sub ions can be retained per metabolite to ensure metabolite analysis flux, thereby reducing the reliability of metabolite identification.

Thermo Scientific ™ Stellar ™ Mass spectrometry fused with Orbitrap Ascend ™ Hyperbolic segmented quadrupole, dual pressure linear ion trap, and Orbitrap Astral ™ The PMT detector has achieved unprecedented scanning speed in linear ion trap mass spectrometry. It pioneered the application of Parallel Reaction Monitoring (PRM) mode from Orbitrap instruments to ion trap mass spectrometry, achieving large-scale metabolomics synchronous qualitative and quantitative analysis, allowing for both accuracy and quantitative reliability in metabolite identification.

Thermo Scientific ™ Stellar ™ mass spectrometer

01

Stellar MS supports non targeted high-resolution data conversion, bridging the "discovery verification" loop

One of the classic strategies in large-scale metabolomics is to convert DDA data collected by high-resolution mass spectrometry into scheduled MRM ion pairs that can be validated by SRM mode, achieving a "discovery validation" loop. Nowadays, this workflow can be easily implemented in the conversion of Orbitrap high-resolution mass spectrometry data to Stellar PRM method, as shown in Figure 1. Compound Discoverer ™ Software and mzVault ™ The software can convert metabolites identified by high-resolution mass spectrometry DDA mode into Stellar ™ The PRM list compatible with mass spectrometry and the secondary mass spectrometry library (. msp) were compiled by TraceFinder ™ Or Skyline ™ The software is used for qualitative and quantitative analysis of metabolites. Skyline ™ The software PRM Conductor function supports automatic optimization of scheduled PRM acquisition channels, reducing the impact of co efflux metabolites on scanning speed. We found that the same plasma samples had extremely high reproducibility in high-resolution mass spectrometry full scan results and Stellar PRM analysis results (screening features with three repeated injections CV<30%), indicating that Stellar mass spectrometry can validate non target discovered metabolic markers at a large-scale sample level.

Figure 1 Development process and result display of Stellar PRM large-scale metabolomics method based on high-resolution mass spectrometry (click to view large image)

02

Stellar MS supports fast transfer using the triple quadrupole method, achieving synchronous qualitative and quantitative analysis

If the user has previously developed targeted or extensively targeted metabolomics analysis methods on the triple quadrupole mass spectrometry platform, Stellar ™ Mass spectrometry can also achieve rapid transfer of existing methods. Under the premise of highly reproducible retention time (same liquid-phase system), Stellar ™ Mass spectrometer can directly convert SRM/MRM list into PRM method; Even if the retention time cannot be reproduced on different chromatographic systems, users can still directly access Stellar ™ On the instrument, rapid library construction is achieved by collecting mixed standards, updating compound retention time and secondary spectra. Unlike traditional SRM/MRM methods, the PRM mode provides a complete secondary spectrum of the analyte, without the need to sacrifice the analysis flux of metabolites to add additional collection channels. This enables synchronous qualitative and quantitative analysis of metabolites, further enhancing confidence in metabolite identification. (Figure 2)

Figure 2. Stellar PRM performs targeted analysis on metabolic features collected by high-resolution mass spectrometry (click to view larger image)

Bile acids are the end products of cholesterol metabolism. Primary bile acids in the human body can be converted by gut microbiota into secondary bile acids with higher biological activity, which play important roles in maintaining cholesterol balance, regulating glucose and lipid metabolism, and inflammatory response in the body. We will use Thermo Scientific previously ™ TSQ Altis ™ The bile acid quantification method constructed on a triple quadrupole mass spectrometer was rapidly transferred to Stellar through the process shown in Figure 3 ™ On MS, synchronous qualitative and quantitative analysis of 33 endogenous bile acids was achieved.

Figure 3. Bile Acid SRM/MRM List PRM Method Transfer and Simultaneous Qualitative and Quantitative Analysis (Click to View Large Image)

03

Stellar MS supports de novo development of targeted lipidomics to achieve precise quantification of triglycerides

Lipids are highly suitable for targeted analysis due to their predictable retention time and secondary mass spectrometry. Skyline ™ The LipidCreator software can generate secondary mass spectra of various lipid adducts, which can be directly used for de novo development of targeted lipidomics methods. Acylcarnitine (AcCar) is one of the key substances in regulating the balance of glucose and lipid metabolism in mammals. It not only helps fatty acids shuttle into mitochondria for beta oxidation, but also participates in the regulation of ketone body production in glucose metabolism. Research has confirmed that acylcarnitine is closely related to various metabolic diseases and has been widely used in the clinical diagnosis of genetic metabolic diseases in newborns. Lipoacylcarnitine has specific fragment ions (m/z 144, m/z 85, m/z 60, as well as neutral loss of specific fatty acid chains), which can be directly predicted using LipidCreator. Using the Stellar PRM model, we identified 207 and 340 acylcarnitines in plasma and urine, respectively, as shown in Figure 4.

Figure 4. Identification of acylcarnitine using Stellar PRM method

(Click to view large image)

04

Stellar MS supports multi-stage mass spectrometry combined scanning to achieve precise analysis of isomers

In lipid analysis, the quantification of triglycerides (TG) with three fatty chains is particularly challenging. For example, when using SRM/MRM mode to quantify TG (50:4), regardless of which fatty chain's neutral loss is used, only the composition of one fatty chain can be determined. For example, using FA16:0 neutral loss fragment (m/z 573.7) for quantification can only prove the existence of FA16:0 in this TG (50:4), but cannot obtain the composition information of the other two fatty acid chains. And Stellar MS3It can accurately locate the three fatty chain compositions of TG, achieving precise quantification of triglycerides (Figure 5).

Figure 5. Stellar tMS3 method for precise quantification of triglycerides

(Click to view large image)

Stellar mass spectrometry does not require separate MS settings3The method can be directly used in parallel with the PRM mode for qualitative and quantitative analysis of metabolites, greatly improving the flexibility of the method. In addition to quantifying triglycerides, MS3It can also be used for accurate quantification of isomers, such as glycerodeoxycholic acid (GCDCA) and glycerodeoxycholic acid (GDCA) in the bile acid family, which can increase MS while quantifying PRM3The channel enhances its quantitative sensitivity and identification accuracy (Figure 6).

Figure 6. Stellar tMS3 Accurate Quantification of Bile Acid Isomers

(Click to view large image)

Large scale metabolomics technology breaks through the limitations of traditional non targeted metabolomics data redundancy and low coverage of targeted analysis. A single experiment can obtain comprehensive screening of thousands of metabolites, while achieving high sensitivity and reproducibility comparable to targeted analysis. Thermo Scientific ™ Stellar ™ The emergence of mass spectrometry marks a revolutionary leap in large-scale metabolomics analysis technology. It combines the excellent quantitative capabilities of quadrupole mass spectrometry with the powerful qualitative capabilities of dual pressure linear ion traps, accelerating the speed of scientific research to clinical application. It greatly promotes the application of metabolomics in large-scale sample analysis scenarios such as clinical queue research and personalized nutritional metabolic profiling, revealing the profound mysteries of life metabolism with more accurate data.

References:

1. Zhou J, Yin Y. Strategies for large-scale targeted metabolomics quanti­cation by liquid chromatography-mass spectrometry. [J] Analyst. 2016; 141(23):6362-6373.

2. Zheng F, Zhao X, Zeng Z, et al. Development of a plasma pseudotargeted metabolomics method based on ultra-high-performance liquid chromatography-mass spectrometry. Nat Protoc. 2020; 15(8):2519-2537.

3. Chiang JY. Bile acids: regulation of synthesis. [J] J Lipid Res. 2009; 50(10): 1955-1966.

4. Di Yu, Lina Zhou, Qiuhui Xuan, et al. Strategy for Comprehensive Identification of Acylcarnitines Based on Liquid Chromatography–High-Resolution Mass Spectrometry. [J] Anal. Chem. 2018, 90(9), 5712–5718.

5. Lauren Bishop, Brittany Lee, Charles Maxey, et al. Combining targeted MS2 and MS3 approaches for the quantitation of bile acids in biological specimens using the Stellar mass spectrometer. [PO]Thermo Fisher Scientific, Inc.

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