Biology subjects

Sulek, K.

Publications and source records attributed to Sulek, K..

3 recordsLinked to original sources

bioRxiv2022

Human cerebrospinal fluid sample preparation and annotation for integrated lipidomics and metabolomics profiling studies

ObjectiveMass spectrometry (MS)-based lipidomics and metabolomics approaches play an essential role in identifying molecular profiles and relevant clinical biomarkers associated with diseases. Cerebrospinal fluid (CSF) is a metabolically diverse biofluid and a key specimen for exploring biochemical changes in neurodegenerative diseases because its composition reflects brain metabolic activity. CSF lipidomics is receiving increasing attention owing to the importance of lipids in brain molecular signaling and their association with several neurological diseases. Detecting lipid species in CSF using MS-based techniques remains challenging because lipids are highly complex in structure and their concentrations span over a broad dynamic range. This work aimed to develop a robust lipidomics and metabolomics method based on commonly used two-phase extraction systems from human CSF samples. MethodsPrioritizing lipid detection, biphasic extraction methods, Folch, Bligh & Dyer (B&D), Matyash and acidified Folch and B&D (aFolch and aB&D), were compared using 150 l of human CSF samples (n=6) for the simultaneous extraction of lipids and metabolites with a wide range of polarity in a single extraction. Multiple chromatographical separation approaches, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), and gas chromatography (GC), were utilized to characterize human CSF metabolome through MS-based untargeted approaches. ResultsA total of 219 lipids across 12 lipid subclasses were identified in CSF samples using RPLC-MS/MS. The aB&D method was found as the most reproducible technique (RSD <15%) for lipid extraction. We found remarkable differences in extraction efficiencies among the five different procedures. The aB&D and B&D yielded the highest peak intensities for targeted lipid internal standards and displayed superior extracting power for major endogenous lipid classes. A total of 674 unique metabolites with a wide polarity range were annotated in CSF using, combining RPLC-MS/MS (n=219), HILIC-MS/MS (n=304) and GC-QTOF MS (n=151). ConclusionsOverall, our findings show that the aB&D extraction method provided suitable lipid coverage, reproducibility, and extraction efficiency for global lipidomics profiling of human CSF samples. In combination with RPLC-MS/MS lipidomics, complementary screening approaches enabled a comprehensive metabolite signature that can be employed in an array of clinical studies.

biochemistry↗

bioRxiv2019

Lysates of Methylococcus capsulatus Bath induce a lean-like microbiota, intestinal FoxP3+RORγt+IL-17+ Tregs and improve metabolism

Interactions between host and gut microbial communities may be modulated by diets and play pivotal roles in securing immunological homeostasis and health. Here we show that intake of feed based on whole-cell lysates of the non-commensal bacterium Methylococcus capsulatus Bath (McB) as protein source reversed high fat high sucrose-induced changes in the gut microbiota to a state resembling that of lean, low fat diet-fed mice, both under mild thermal stress (T22{degrees}C) and at thermoneutrality (T30{degrees}C). McB feeding selectively upregulated triple positive (Foxp3+ROR{gamma}t+IL-17+) regulatory T cells in the small intestine and colon, and enhanced mucus production and glycosylation status suggesting improved gut health. Mice receiving McB lysates further exhibited improved glucose regulation, reduced body and liver fat along with diminished hepatic immune infiltration. Collectively, these data points towards profound whole-body effects elicited by the McB lysate suggesting that it may serve as a potent modulator of immunometabolic homeostasis.

immunology↗

bioRxiv2019

Trapped ion mobility spectrometry (TIMS) and parallel accumulation - serial fragmentation (PASEF) enable in-depth lipidomics from minimal sample amounts

Lipids form a highly diverse group of biomolecules fulfilling central biological functions, ranging from structural components to intercellular signaling. Yet, a comprehensive characterization of the lipidome from limited starting material, for example in tissue biopsies, remains very challenging. Here, we develop a high-sensitivity lipidomics workflow based on nanoflow liquid chromatography and trapped ion mobility spectrometry. Taking advantage of the PASEF principle (Meier et al., PMID: 26538118), we fragmented on average nine precursors in each 100 ms TIMS scans, while maintaining the full mobility resolution of co-eluting isomers. The very high acquisition speed of about 100 Hz allowed us to obtain MS/MS spectra of the vast majority of detected isotope patterns for automated lipid identification. Analyzing 1 uL of human plasma, PASEF almost doubled the number of identified lipids over standard TIMS-MS/MS and allowed us to reduce the analysis time by a factor of three without loss of coverage. Our single-extraction workflow surpasses the plasma lipid coverage of extensive multi-step protocols in common lipid classes and achieves attomole sensitivity. Building on the high precision and accuracy of TIMS collisional cross section measurements (median CV 0.2%), we compiled 1,327 lipid CCS values from human plasma, mouse liver and human cancer cells. Our study establishes PASEF in lipid analysis and paves the way for sensitive, ion mobility-enhanced lipidomics in four dimensions.

systems biology↗