Concentric Hybrid Nanoelectrospray Ionization-Atmospheric Pressure Chemical Ionization Source for High-Coverage Mass Spectrometry Analysis of Single-Cell Metabolomics.

High-coverage mass spectrometry analysis of single-cell metabolomics remains challenging due to the extremely low abundance and wide polarity of metabolites and ultra-small volume in single cells. Herein, a novel concentric hybrid ionization source, nanoelectrospray ionization-atmospheric pressure chemical ionization (nanoESI-APCI), is ingeniously designed to detect polar and nonpolar metabolites simultaneously in single cells. The source is constructed by inserting a pulled glass capillary coaxially into a glass tube that acts as a dielectric barrier layer. Benefitting from the integrated advantages of nanoESI and APCI, its limit of detection is improved by one order of magnitude to 10 pg mL-1. After the operational parameter optimization, 254 metabolites detected in nanoESI-APCI are tentatively identified from a single cell, and 82 more than those in nanoESI. The developed nanoESI-APCI is successively applied to study the metabolic heterogeneity of human hepatocellular carcinoma tissue microenvironment united with laser capture microdissection (LCM), the discrimination of cancer cell types and subtypes, the metabolic perturbations to glucose starvation in MCF7 cells and the metabolic regulation of cancer stem cells. These results demonstrated that the nanoESI-APCI not only opens a new avenue for high-coverage and high-sensitivity metabolomics analysis of single cell, but also facilitates spatially resolved metabolomics study coupled with LCM.

Effect of roxadustat on serum metabolome and lipidome in patients with end-stage renal disease and erythropoiesis-stimulating agent resistance.

Background: Roxadustat is a newly marketed hypoxia-inducible factor prolyl hydroxylase inhibitor used to treat anemia in patients with end-stage renal disease (ESRD). While clinical trials have demonstrated the therapeutic effects of roxadustat in patients with ESRD who are resistant to erythropoiesis-stimulating agents (ESAs), its metabolic effects are still unclear. Methods: Thirty-two individuals with ESRD and ESA resistance from the Blood Purification Center of Dalian Municipal Central Hospital were included. A total of 96 fasting serum samples were obtained from participants before treatment with roxadustat, and after treatment for 15 and 30 days. Ultra-high performance liquid chromatography-mass spectrometry-based metabolomics and lipidomics strategies were applied to investigate the effects of roxadustat on serum metabolism. Results: A total of 255 metabolites and 444 lipid molecular species were detected and quantified. Sphingolipids and phospholipids decreased significantly during treatment, possibly associated with changes in phospholipid and ceramide metabolism. Bile acid levels decreased and cholic acid/chenodeoxycholic acid increased, indicating changes in gut microbiota and bile acid metabolism. Amino acids also changed during the process of treatment. Conclusions: The present study showed sphingolipids, phospholipids, and bile acids were significantly altered, which may be associated with a changed metabolism caused by roxadustat. This approach provided a powerful tool for exploring the mechanisms of ESA resistance in ESRD patients and may represent a promising strategy for elucidating the complex therapeutic mechanisms of other drugs.

High-throughput single cell metabolomics and cellular heterogeneity exploration by inertial microfluidics coupled with pulsed electric field-induced electrospray ionization-high resolution mass spectrometry.

Single cell metabolomics can obtain the metabolic profiles of individual cells and reveal cellular heterogeneity. However, high-throughput single-cell mass spectrometry (MS) analysis under physiological conditions remains a great challenge due to the presence of complex matrix and extremely small cell volumes. Herein, a serpentine channel microfluidic device which was designed to achieve continuous cell separation and inertial focusing, was coupled with a pulsed electric field-induced electrospray ionization-high resolution MS (PEF-ESI-HRMS) to achieve high-throughput single cell analysis. The pulsed square wave electric field was applied to realize on-line cell disruption and induce electrospray ionization. Single cells were analyzed under near-physiological conditions at a throughput of up to 80 cells min-1. More than 900 features were detected and approximately 120 metabolites were tentatively identified from a single cell. Further, by continually analyzing more than 3000 MCF7 and HepG2 cells, discrimination of different cancer cells based on their individual metabolic profiles was achieved by using the principal component analysis. The PEF-ESI-HRMS method was also applied for the analysis of single yeast cells, and more than 40 metabolites were annotated. This method is versatile and has good robustness, which is promising for high-throughput single cell metabolomics analysis.

Lipid Profiling of 20 Mammalian Cells by Capillary Microsampling Combined with High-Resolution Spectral Stitching Nanoelectrospray Ionization Direct-Infusion Mass Spectrometry.

Studies of cellular metabolism can provide profound insights into the underlying molecular mechanisms and metabolic function. To date, the majority of cellular metabolism studies based on chromatography-mass spectrometry (MS) require population cells to obtain informative metabolome. These methods are not only time-consuming but also not suitable for amount-limited cell samples such as circulating tumor cells, stem cells, and neurons. Therefore, it is extremely essential to develop analytical methods enabling to detect metabolome from tens of cells in a high-throughput and high-sensitivity way. In this work, a novel platform for rapid and sensitive detection of lipidome in 20 mammalian cells was proposed using capillary microsampling combined with high-resolution spectral stitching nanoelectrospray ionization direct-infusion MS. It can be used to collect cells rapidly and accurately via the capillary microprobe, extract lipids directly in a 96-well plate using a spray solvent, and detect more than 500 lipids covering 19 lipid subclasses within 3 min. This novel platform was successfully applied to study the lipid features of different cancer cell types and subtypes as well as target cells from tissue samples. This study provides a strategy for determining the lipid species with rich information in tens of cells and demonstrates great potential for clinical applications.

Strategy for Nontargeted Metabolomic Annotation and Quantitation Using a High-Resolution Spectral-Stitching Nanoelectrospray Direct-Infusion Mass Spectrometry with Data-Independent Acquisition.

Direct-infusion nanoelectrospray ionization high-resolution mass spectrometry (DI-nESI-HRMS) is an alternative approach to chromatography-MS-based techniques for nontargeted metabolomics, offering a high sample throughout. However, its annotation accuracy of analytes is still full of challenges. In this study, we proposed a strategy for the annotation and quantitation of nontargeted metabolomic data using a spectral-stitching DI-nESI-HRMS with data-independent acquisition. The metabolite annotation strategy included the isotopic distribution, MS/MS spectrum similarity, and precursor and product ion correlation as well as matching of the extracted metabolite features along with the targeted metabolite precursors. Two groups of mixed standard solutions containing 40 and 79 metabolites were, respectively, used to establish the metabolite annotation strategy and validate its reliability. The results showed that the detected standards could be well annotated at top three explanations and total qualitative percentages were 100% (40 of 40) for the standard solution and 94.9% (74 of 78) for the standards spiked into the serum matrix. The intensity of the precursor ions was used for quantitation except for isomers, which were quantified by the intensities of the characteristic product ions if available. Finally, the strategy was applied to study serum metabolomics in diabetes, and the results demonstrated that it is promising for a large-scale cohort metabolomic study.

A high throughput lipidomics method and its application in atrial fibrillation based on 96-well plate pretreatment and liquid chromatography-mass spectrometry.

Successful applications of lipidomics in clinic need study large-scale samples, and the bottlenecks are in throughput and robustness of the lipid analytical method. Here, we report an untargeted lipidomics method by combining high throughput pretreatment in the 96-well plate with ultra-high performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry. The developed method was validated to have satisfactory analytical characteristics in terms of linearity, repeatability and extraction recovery. It can be used to handle 96 samples simultaneously in 25 min and detect 441 lipids in plasma sample. Storage stability investigation on lipid extracts provided an operable procedure for large-scale sample analysis and demonstrated most lipids were stable in autosampler at 10 °C within 36 h and at -80 °C within 72 h after the pretreatment. To prove the usefulness, the method was employed to investigate abnormal plasma lipidome related to atrial fibrillation. A biomarker panel with the area under the curve (AUC) values of 0.831 and 0.745 was achieved in the discovery and external validation sets, respectively. These results showed that the developed method is applicable for large-scale biological sample handling and lipid analysis of plasma.

Recent advances in analytical strategies for mass spectrometry-based lipidomics.

Lipids are vital biological molecules and play multiple roles in cellular function of mammalian organisms such as cellular membrane anchoring, signal transduction, material trafficking and energy storage. Driven by the biological significance of lipids, lipidomics has become an emerging science in the field of omics. Lipidome in biological systems consists of hundreds of thousands of individual lipid molecules that possess complex structures, multiple categories, and diverse physicochemical properties assembled by different combinations of polar headgroups and hydrophobic fatty acyl chains. Such structural complexity poses a huge challenge for comprehensive lipidome analysis. Thanks to the great innovations in chromatographic separation techniques and the continuous advances in mass spectrometric detection tools, analytical strategies for lipidomics have been highly diversified so that the depth and breadth of lipidomics have been greatly enhanced. This review will present the current state of mass spectrometry-based analytical strategies including untargeted, targeted and pseudotargeted lipidomics. Recent typical applications of lipidomics in biomarker discovery, pathogenic mechanism and therapeutic strategy are summarized, and the challenges facing to the field of lipidomics are also discussed.