Ion-Pair Selection Method for Pseudotargeted Metabolomics Based on SWATH MS Acquisition and Its Application in Differential Metabolite Discovery of Type 2 Diabetes

Abstract

The pseudotargeted metabolomics method integrates advantages of nontargeted and targeted analysis because it can acquire data of metabolites in the multireaction monitoring (MRM) mode of mass spectrometry (MS) without needing standards. The key is the ion-pair information collection from samples to be analyzed. It is well-known that sequential windowed acquisition of all theoretical Fragment ion (SWATH) MS mode can acquire MS2 information to a maximum extent. To expediently acquire as many ion-pairs as possible with optimal collision energy (CE), an ion-pair selection approach based on SWATH MS acquisition with variable isolation windows was developed in this study. Initially, nontargeted acquisition of all metabolites information in plasma Standard Reference Material (SRM 1950) was performed by ultra high-performance liquid chromatography (UHPLC)-quadrupole time-of-flight (Q-TOF) MS platform with three CEs. With the help of software tool, the ion-pairs of unique metabolites were gained. Then they were validated in scheduled MRM coupled with UHPLC. After removing false positive, the ion-pairs with an optimal CE was integrated. A total of 1373 unique metabolite ion-pairs were obtained at positive ion mode. And repeatability of the established pseudotargeted approach was evaluated by intraday and interday precision. The results demonstrated the method was stable, reliable, and suitable for metabolomics study. As an application example, alterations of serum metabolites in Type 2 diabetes were investigated by using the established method. This work provides a pseudotargeted ion-pair selection method based on SWATH MS acquisition with the characters of increased metabolite coverage, suitable CE, and convenient processing.

Bridging Targeted and Untargeted Mass Spectrometry-Based Metabolomics via Hybrid Approaches

“…Over the past few years, hybrid approaches in metabolomics have been applied in biological samples that are associated with different kinds of disease, such as cardiovascular disease [ 76 ], neurodegenerative disease [ 77 ], cancer [ 39 , 40 , 41 ], kidney dysfunction [ 78 ], and diabetes mellitus [ 55 ]. The established hybrid methods discussed above also have been used in a variety of biological samples (such as urine, serum/plasma, and tissue samples) to facilitate broader metabolite coverage or to validate metabolite biomarkers for disease.…”

“…In addition to cancer research, a pseudotargeted method based on UHPLC−MS with SWATH acquisition was also applied to a Type 2 diabetes study. In this study [ 55 ], 162 significantly changed metabolites were defined in the serum of type 2 diabetes patients, which demonstrated that the pseudotargeted method can provide many useful hints for investigating changes in metabolites and has great potential in promoting our understanding of the metabolism processes of metabolic disease.…”

Enhanced pseudotargeted analysis using a segment data dependent acquisition strategy by liquid chromatography–tandem mass spectrometry for a metabolomics study of liquiritin in the treatment of depression

“…Wang et al. [17] proposed a sequential windowed acquisition of all theoretical fragment ion strategy to improve the acquisition of MS/MS spectra, especially for low abundant metabolites. Although these attempts have made some improvements in the coverage of metabolites, there are some drawbacks, e.g.…”

A combined targeted/untargeted LC-MS/MS-based screening approach for mammalian cell lines treated with ionic liquids: Toxicity correlates with metabolic profile

“…DDA methods use information from an MS scan for selection of the most abundant precursors for fragmentation being able to identify metabolites based on MS/MS spectra. Unfortunately, low abundant metabolites are not triggered for fragmentation [11,13,[17][18][19][20]. Relative quantification is exclusively based on MS data which may be less sensitive.…”

“…Consequently, qualitative analysis of unknowns may be quite challenging by these classical DIA methods [20,22]. It has been shown that a better spectral quality data can be obtained with a DIA method called sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) [17,19], an acquisition approach first proposed for linear ion trap [23]. In DIA with SWATH, a sequence of MS/MS experiments with intermediate wide Q1 precursor ion selection windows (SWATH windows) (typically 20-30 Da wide) are consecutively stepped through the targeted m/z range [11,22].…”