Pseudotargeted Metabolomics Method and Its Application in Serum Biomarker Discovery for Hepatocellular Carcinoma Based on Ultra High-Performance Liquid Chromatography/Triple Quadrupole Mass Spectrometry

Abstract

Untargeted analysis performed using full-scan mass spectrometry (MS) coupled with liquid chromatography (LC) is commonly used in metabolomics. Although they are commonly employed, full-scan MS methods such as quadrupole-time-of-flight (Q-TOF) MS have been restricted by various factors including their limited linear range and complicated data processing. LC coupled with triple quadrupole (QQQ) MS operated in the multiple reaction monitoring (MRM) mode is the gold standard for metabolite quantification; however, only known metabolites are generally quantified, limiting its applications in metabolomic analysis. In this study, a pseudotargeted approach was proposed to perform serum metabolomic analysis using an ultra high-performance liquid chromatography (UHPLC)/QQQ MS system operated in the MRM mode, for which the MRM ion pairs were acquired from the serum samples through untargeted tandem MS using UHPLC/Q-TOF MS. The UHPLC/QQQ MRM MS-based pseudotargeted method displayed better repeatability and wider linear range than the traditional UHPLC/Q-TOF MS-based untargeted metabolomics method, and no complicated peak alignment was required. The developed method was applied to discover serum biomarkers for patients with hepatocellular carcinoma (HCC). Patients with HCC had decreased lysophosphatidylcholine, increased long-chain and decreased medium-chain acylcarnitines, and increased aromatic and decreased branched-chain amino acid levels compared to healthy controls. The novelty of this work is that it provides an approach to acquire MRM ion pairs from real samples, is not limited to metabolite standards, and it provides a foundation to achieve pseudotargeted metabolomic analysis on the widely used LC/QQQ MS platform.

Globally Optimized Targeted Mass Spectrometry: Reliable Metabolomics Analysis with Broad Coverage

“…This improved performance results primarily from the fact that GOT-MS is based on the QQQ-MS platform, which was also observed in previous studies. 35,36 In addition, while our Q-TOF generally has good reproducibility for strong signals, these signals cannot be too large, because signal saturation will deteriorate mass accuracy (as well as linearity). Therefore, the advantages of high resolution and high mass accuracy can be compromised in traditional global profiling.…”

UPLC-based metabonomic applications for discovering biomarkers of diseases in clinical chemistry

“…In addition, UPLC-QTOF/MS and UPLC-MS/MS approaches were used for qualitative and quantitative analyses of serum biomarkers for patients with HCC. The results indicated that patients with HCC had decreased LPCs, increased long-chain and decreased medium-chain acylcarnitines, and increased aromatic and decreased branched-chain amino acid [30]. UPLC-QTOF/MS and UPLC triple quadrupole linear ion trap MS approaches were performed on qualitative and quantitative comparisons of metabolite levels in sera of HCC patients and cirrhosis patients from Egypt [31].…”

An untargeted metabolomics-driven approach based on LC–TOF/MS and LC–MS/MS for the screening of xenobiotics and metabolites of Zhi-Zi-Da-Huang decoction in rat plasma

“…Therefore, an unbiased and robust discrimination method should be developed to enhance the efficiency of analytical processes in xenobiotic metabolism research. By measuring and mathematically modelling changes in the levels of products of metabolism found in biological fluids and tissues [10][11][12], untargeted metabolomics provides fresh insight into subtle changes of endogenous components [13][14][15], and also could be extended to xenobiotics and related metabolites to which we are exposed. Applying untargeted metabolomicsdriven approach, the interesting variables could be extracted easily from the xenobiotic metabolome without the need for priori knowledge of the compound structure.…”