Abstract
IntroductionRecent advances in high-throughput methodologies in the ‘omics’ and synthetic biology fields call for rapid and sensitive workflows in the metabolic phenotyping of complex biological samples.ObjectiveThe objective of this research was to evaluate a straightforward to implement LC–MS metabolomics method using a commercially available chromatography column that provides increased throughput. Reducing run time can potentially impact chromatography and therefore the effects of ion mobility spectrometry to expand peak capacity were also evaluated. Additional confidence provided via collision cross section measurements for detected features was also explored.MethodsA rapid untargeted metabolomics workflow was developed with broad metabolome coverage, combining zwitterionic-phase hydrophilic interaction chromatography (HILIC-Z) with drift tube ion mobility-quadrupole time-of-flight (DTIM-qTOF) mass spectrometry. The analytical performance of our method was explored using extracts from complex biological samples, including a reproducibility study on chicken serum and a simple comparative study on a bacterial metabolome.ResultsThe method is acronymised RHIMMS for rapid HILIC-Z ion mobility mass spectrometry. We present the RHIMMS workflow starting with data acquisition, followed by data processing and analysis. RHIMMS demonstrates improved chromatographic separation for a selection of metabolites with wide physicochemical properties while maintaining reproducibility at better than 20% over 200 injections at 3.5 min per sample for the selected metabolites, and a mean of 13.9% for the top 50 metabolites by intensity. Additionally, the combination of rapid chromatographic separation with ion mobility allows improved annotation and the ability to distinguish isobaric compounds.ConclusionOur results demonstrate RHIMMS to be a rapid, reproducible, sensitive and high-resolution analytical platform that is highly applicable to the untargeted metabolomics analysis of complex samples.
Highlights
Recent advances in high-throughput methodologies in the ‘omics’ and synthetic biology fields call for rapid and sensitive workflows in the metabolic phenotyping of complex biological samples
Rapid HILIC‐Z ion mobility mass spectrometry (RHIMMS) metabolomics analysis follows the typical workflow of metabolomics studies, starting with biological sample preparation, continuing with data acquisition, data processing, statistical analysis, and culminating in data interpretation (Fig. 1)
We evaluated RHIMMS, a rapid LC-IM-MS method utilizing a short HILIC-Z column in combination with DTIMqTOF to assess its suitability for routine metabolomics in a high throughput laboratory
Summary
Recent advances in high-throughput methodologies in the ‘omics’ and synthetic biology fields call for rapid and sensitive workflows in the metabolic phenotyping of complex biological samples. Conclusion Our results demonstrate RHIMMS to be a rapid, reproducible, sensitive and high-resolution analytical platform that is highly applicable to the untargeted metabolomics analysis of complex samples. Incorporating ion mobility spectrometry (IMS) into LC–MS-based metabolomics methodologies enables structural evaluation of small molecules in complex matrices by assessing the drift times (tD) and collision cross section (CCS) values for individual features (via IMS), in addition to chromatographic retention times (via LC) and m/z values (via MS); these parameters combined provide increased resolution and more accurate identification of small molecules present in complex biological and environmental samples (Gabelica & Marklund, 2018; Gika et al, 2019; Odenkirk & Baker, 2020)
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