Simultaneous Quantitation and Discovery (SQUAD) Analysis: Combining the Best of Targeted and Untargeted Mass Spectrometry-Based Metabolomics

Abstract

Untargeted and targeted approaches are the traditional metabolomics workflows acquired for a wider understanding of the metabolome under focus. Both approaches have their strengths and weaknesses. The untargeted, for example, is maximizing the detection and accurate identification of thousands of metabolites, while the targeted is maximizing the linear dynamic range and quantification sensitivity. These workflows, however, are acquired separately, so researchers compromise either a low-accuracy overview of total molecular changes (i.e., untargeted analysis) or a detailed yet blinkered snapshot of a selected group of metabolites (i.e., targeted analysis) by selecting one of the workflows over the other. In this review, we present a novel single injection simultaneous quantitation and discovery (SQUAD) metabolomics that combines targeted and untargeted workflows. It is used to identify and accurately quantify a targeted set of metabolites. It also allows data retro-mining to look for global metabolic changes that were not part of the original focus. This offers a way to strike the balance between targeted and untargeted approaches in one single experiment and address the two approaches’ limitations. This simultaneous acquisition of hypothesis-led and discovery-led datasets allows scientists to gain more knowledge about biological systems in a single experiment.

High-Resolution Mass Spectrometry for Human Exposomics: Expanding Chemical Space Coverage

“…Jia and colleagues (2019) integrated targeted and non-targeted methods for exposomics of human urine by isotopically tagging exposure biomarkers with common functional groups (e.g., phenolic, hydroxyl, carboxyl, and primary amine) that can simultaneously be leveraged for quantitative purposes . Likewise, the single-injection s imultaneous q uantitation a nd d iscovery (SQUAD) approach, developed for metabolomics, identifies (via ASDs) and quantifies (via calibration curves or one-point calibration using ASDs and ISDs) select compounds while allowing simultaneous data mining to look for broader molecular changes . Second, the feasibility would largely depend on the specific instrumental capacity in use.…”

Antiviral Mechanisms of Anisomycin Produced by Streptomyces albulus SN40 on Potato Virus Y

“…20,21 Specifically, targeted metabolomics is the validation and analysis of specific metabolites, while untargeted metabolomics is a comprehensive and systematic analysis of the organism's metabolites. 22 In this study, we isolated a new Streptomyces strain SN40 whose fermentation filtrate can effectively suppress the infection of TMV and PVY in plants. Physiological, biochemical classification and phylogenetic analysis showed that the Streptomyces strain SN40 is most closely related to Streptomyces albulus.…”

“…Metabolomics has been widely used in the fields of agriculture, medicine, pharmacy, and food chemistry, which is generally based on nuclear magnetic resonance (NMR), and gas chromatography (GC) or liquid chromatography (LC) coupled to mass spectrometry (MS) analytical techniques. , Specifically, targeted metabolomics is the validation and analysis of specific metabolites, while untargeted metabolomics is a comprehensive and systematic analysis of the organism’s metabolites …”

Uniting targeted (Zeno MRM-HR) and untargeted (SWATH) LC-MS in a single run for sensitive high-resolution exposomics.

“…As future directions, the authors highlight the efforts needed towards the combination of targeted and untargeted metabolomics approaches via untargeted MS1 data acquisition and high-resolution MRM transitions for targeting multiple metabolites. More recently, Bird et al (2023) 10 have introduced a new workflow based on orbitrap and/or TOF systems (SQUAD, Simultaneous Quantitation and Discovery Analysis) aiming at combining both targeted and untargeted with the main objective of finding the equilibrium between untargeted and targeted approaches in a single experiment. This paper describes, to the best of our knowledge, the first targeted/untargeted, single-injection LC-MS method for integrated exposomics and metabolomics, combining the advantages of both approaches, i.e., ultrahigh sensitivity (targeted) and broad coverage and retrospective data analysis (untargeted).…”