A reversed phase ultra-high-performance liquid chromatography-data independent mass spectrometry method for the rapid identification of mycobacterial lipids

Sakallioglu, Isin T.; Maroli, Amith S.; Leite, Aline de Lima; Powers, Robert

doi:10.1016/j.chroma.2021.462739

Cited by 5 publications

(7 citation statements)

References 48 publications

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“…We employed our previously described LC−MS lipidomics protocol to maximize the coverage of the Msm lipidome to include mycolic acids. 49 The same cellular extract samples were used for both the metabolomics and lipidomics experiments. The LC−MS data set was collected in both negative and positive ionization modes, which yielded 6,033 and 5,281 spectral features, respectively.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Msm cells were treated with DA-CB or DMSO (as a control) and lysed, and the metabolome, lipidome, and proteome were extracted for reversed-phase ultra-high-pressure liquid chromatography mass spectrometry in a data-independent acquisition mode (RP UHPLC-DIA-MS). The RP UHPLC-DIA-MS lipidomics data acquisition protocol was previously optimized to ensure complete coverage of the mycobacterial lipidome, with a special emphasis in the detection of mycolic acids . The lipidomics and metabolomics data were processed and analyzed with statistical software packages .…”

Section: Resultsmentioning

confidence: 99%

“…The RP UHPLC-DIA-MS lipidomics data acquisition protocol was previously optimized to ensure complete coverage of the mycobacterial lipidome, with a special emphasis in the detection of mycolic acids. 49 The lipidomics and metabolomics data were processed and analyzed with statistical software packages. 50 The multi-omics protocol employed to characterize the cellular impact of DA-CB is summarized in Scheme 1.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…We characterized the lipidome changes to further explore the impact of DA-CB on Msm metabolism, especially given the predicted relationship between DA-CB and the mycolic acid inhibitor ETH. We employed our previously described LC–MS lipidomics protocol to maximize the coverage of the Msm lipidome to include mycolic acids . The same cellular extract samples were used for both the metabolomics and lipidomics experiments.…”

Section: Resultsmentioning

confidence: 99%

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Multi-omics Investigation into the Mechanism of Action of an Anti-tubercular Fatty Acid Analogue

et al. 2022

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The mechanism of action (MoA) of a clickable fatty acid analogue 8-(2-cyclobuten-1-yl)octanoic acid (DA-CB) has been investigated for the first time. Proteomics, metabolomics, and lipidomics were combined with a network analysis to investigate the MoA of DA-CB against Mycobacterium smegmatis (Msm). The metabolomics results showed that DA-CB has a general MoA related to that of ethionamide (ETH), a mycolic acid inhibitor that targets enoyl-ACP reductase (InhA), but DA-CB likely inhibits a step downstream from InhA. Our combined multi-omics approach showed that DA-CB appears to disrupt the pathway leading to the biosynthesis of mycolic acids, an essential mycobacterial fatty acid for both Msm and Mycobacterium tuberculosis (Mtb). DA-CB decreased keto-meromycolic acid biosynthesis. This intermediate is essential in the formation of mature mycolic acid, which is a key component of the mycobacterial cell wall in a process that is catalyzed by the essential polyketide synthase Pks13 and the associated ligase FadD32. The multi-omics analysis revealed further collateral alterations in bacterial metabolism, including the overproduction of shorter carbon chain hydroxy fatty acids and branched chain fatty acids, alterations in pyrimidine metabolism, and a predominate downregulation of proteins involved in fatty acid biosynthesis. Overall, the results with DA-CB suggest the exploration of this and related compounds as a new class of tuberculosis (TB) therapeutics. Furthermore, the clickable nature of DA-CB may be leveraged to trace the cellular fate of the modified fatty acid or any derived metabolite or biosynthetic intermediate.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Multi-omics Investigation into the Mechanism of Action of an Anti-tubercular Fatty Acid Analogue

et al. 2022

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“…columns, and was successfully evaluated and implemented in proteomics , and metabolomics , studies. Previous studies have employed microbore separation to pursue lipidomics. − However, a study collectively presenting microbore separation method optimization is needed as a reproducibility assessment, with benchmarking against a standard narrow-bore column.…”

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confidence: 99%

High-Throughput Microbore LC-MS Lipidomics to Investigate APOE Phenotypes

Gadara,

Berka,

Spacil

2023

Anal. Chem.

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Microflow liquid chromatography interfaced with mass spectrometry (μLC-MS/MS) is increasingly applied for high-throughput profiling of biological samples and has been proven to have an acceptable trade-off between sensitivity and reproducibility. However, lipidomics applications are scarce. We optimized a μLC-MS/MS system utilizing a 1 mm inner diameter × 100 mm column coupled to a triple quadrupole mass spectrometer to establish a sensitive, high-throughput, and robust single-shot lipidomics workflow. Compared to conventional lipidomics methods, we achieve a ∼4-fold increase in response, facilitating quantification of 351 lipid species from a single iPSC-derived cerebral organoid during a 15 min LC-MS analysis. Consecutively, we injected 303 samples over ∼75 h to prove the robustness and reproducibility of the microflow separation. As a proof of concept, μLC-MS/MS analysis of Alzheimer’s disease patient-derived iPSC cerebral organoid reveals differential lipid metabolism depending on APOE phenotype (E3/3 vs E4/4). Microflow separation proves to be an environmentally friendly and cost-effective method as it reduces the consumption of harmful solvents. Also, the data demonstrate robust, in-depth, high-throughput performance to enable routine clinical or biomedical applications.

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Multiplatform untargeted metabolomics

Jeppesen

Powers

2023

Magnetic Reson in Chemistry

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Metabolomics samples like human urine or serum contain upwards of a few thousand metabolites, but individual analytical techniques can only characterize a few hundred metabolites at best. The uncertainty in metabolite identification commonly encountered in untargeted metabolomics adds to this low coverage problem. A multiplatform (multiple analytical techniques) approach can improve upon the number of metabolites reliably detected and correctly assigned. This can be further improved by applying synergistic sample preparation along with the use of combinatorial or sequential non‐destructive and destructive techniques. Similarly, peak detection and metabolite identification strategies that employ multiple probabilistic approaches have led to better annotation decisions. Applying these techniques also addresses the issues of reproducibility found in single platform methods. Nevertheless, the analysis of large data sets from disparate analytical techniques presents unique challenges. While the general data processing workflow is similar across multiple platforms, many software packages are only fully capable of processing data types from a single analytical instrument. Traditional statistical methods such as principal component analysis were not designed to handle multiple, distinct data sets. Instead, multivariate analysis requires multiblock or other model types for understanding the contribution from multiple instruments. This review summarizes the advantages, limitations, and recent achievements of a multiplatform approach to untargeted metabolomics.

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A reversed phase ultra-high-performance liquid chromatography-data independent mass spectrometry method for the rapid identification of mycobacterial lipids

Cited by 5 publications

References 48 publications

Multi-omics Investigation into the Mechanism of Action of an Anti-tubercular Fatty Acid Analogue

Multi-omics Investigation into the Mechanism of Action of an Anti-tubercular Fatty Acid Analogue

High-Throughput Microbore LC-MS Lipidomics to Investigate APOE Phenotypes

Multiplatform untargeted metabolomics

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