Biology subjects

Holcapek, M.

Publications and source records attributed to Holcapek, M..

4 recordsLinked to original sources

bioRxiv2025

Comprehensive Lipidomic Characterization of Human Blood by High-Throughput UHPSFC/MS using Bioinert Column and Modified MTBE Extraction

Supercritical fluid chromatography is traditionally employed for nonpolar and moderately polar analytes, while the analysis of ionic compounds remains a recognized limitation of the technique. Here, we introduce a novel ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method using a bioinert column, enabling the separation of lipids with a broad polarity range from nonpolar to ionic species. The UHPSFC/MS method was optimized using 79 lipid species across 41 lipid subclasses, achieving a total run time of 7.5 min, including the column equilibration. The comparison of the separation with conventional and bioinert columns revealed a substantial improvement in peak shapes for ionic lipid classes, such as PS, LPS, PA, LPA, CerP, and SPBP. Additionally, we introduce a combination of the modified chloroform-free extraction followed by a hexane elimination step, which reduces concentrations of nonpolar lipids allowing the injection of more concentrated extracts and minimize ion source contamination. The optimized methodology was applied for the untargeted analysis of human plasma and erythrocyte-rich fraction to achieve highly confident identification of 657 lipid species across 37 lipid subclasses in human blood. The method followed the recommendations for validation of (bio)analytical methods, and its accuracy was confirmed by quantitative analysis of the reference material NIST SRM 1950, with the determined concentrations in agreement with the consensus values from ring trials. The current methodology represents a novel high-throughput and comprehensive quantitative lipidomic method for biological samples. Moreover, the findings provide the potential for the development of bioinert components for SFC platforms.

biochemistry↗

bioRxiv2025

LipidQMap - An Open-Source Tool for Quantitative Mass Spectrometry Imaging of Lipids

Mass spectrometry imaging (MSI) is a powerful tool in both basic and clinical research, enabling spatial visualization of biomolecules and drugs in tissue sections. However, factors influencing mass spectrometric response during imaging are increasingly recognized for their impact on apparent molecular distribution. Quantitative mass spectrometry imaging (qMSI) addresses this variability by incorporating analytical standards that undergo the same processes as analytes. While qMSI sample preparation protocols for omics-scale quantitative lipidomics are actively evolving, software solutions for downstream data processing remain scarce. Here, we introduce LipidQMap, the first open-source platform for processing omics-scale qMSI lipidomics data. LipidQMap applies one-point calibration, normalizing annotated lipid signals against class-specific standards on a pixel-by-pixel basis, and generates concentration heat maps in pmol/mm2. The software supports centroided data import, recalibration, and lipid identification using a built-in, user-modifiable lipid database. LipidQMap resolves Na/H adduct isobaric overlaps by leveraging sodiated-to-protonated adduct ratios of standards, validated across several MSI platforms using mouse brain sections. Type II isobaric overlaps are corrected using predicted isotopic patterns. Extensive validation demonstrates that LipidQMap is robust across MSI platforms and harmonizes qMSI data, enabling more accurate and reproducible spatial lipid quantification.

bioinformatics↗

bioRxiv2022

Altered plasma membrane abundance of the sulfatide-binding protein NF155 links glycosphingolipid imbalances to demyelination

Myelin is a multi-layered membrane that tightly wraps neuronal axons enabling efficient, high-speed signal propagation. The axon and myelin sheath form tight contacts, mediated by specific plasma membrane proteins and lipids, and disruption of these contacts causes devastating demyelinating diseases. Using two cell-based models of demyelinating sphingolipidoses, we demonstrate that altered lipid metabolism changes the abundance of specific plasma membrane proteins. These altered membrane proteins have known roles in cell adhesion and signalling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule Neurofascin, a protein critical for the maintenance of myelin-axon contacts, changes following disruption to sphingolipid metabolism. This provides a direct molecular link between altered lipid abundance and myelin stability. We show that the Neurofascin isoform NF155, but not NF186, interacts directly and specifically with the sphingolipid sulfatide via multiple binding sites and that this interaction requires the full-length extracellular domain of NF155. We demonstrate that NF155 adopts an S-shaped conformation and preferrentially binds sulfatide-containing membranes in cis, with important implications for protein arrangement in the tight axon-myelin space. Our work links glycosphingolipid imbalances to disturbance of membrane protein abundance and demonstrates how this may be driven by direct protein-lipid interactions, providing a mechanistic framework to understand the pathogenesis of galactosphingolipidoses.

neuroscience↗

bioRxiv2021

Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS

Reversed-phase ultrahigh-performance liquid chromatography q mass spectrometry (RP-UHPLC/MS) method was developed with the aim to unambiguously identify a large number of lipid species from multiple lipid classes in human plasma. The optimized RP-UHPLC/MS method employed the C18 column with sub-2 m particles with the total run time of 25 min. The chromatographic resolution was investigated with 42 standards from 18 lipid classes. The UHPLC system was coupled to high-resolution quadrupole - time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) measuring full scan and tandem mass spectra (MS/MS) in positive- and negative-ion modes with high mass accuracy. Our identification approach was based on m/z values measured with mass accuracy within 5 ppm tolerance in the full scan mode, characteristic fragment ions in MS/MS, and regularity in chromatographic retention dependences for individual lipid species, which provides the highest level of confidence for reported identifications of lipid species including regioisomeric and other isobaric forms. The graphs of dependences of retention times on the carbon number or on the number of double bond(s) in fatty acyl chains were constructed to support the identification of lipid species in homologous lipid series. Our list of identified lipid species is also compared with previous publications investigating human blood samples by various MS based approaches. In total, we have reported more than 500 lipid species representing 26 polar and nonpolar lipid classes detected in NIST Standard reference material 1950 human plasma.

biochemistry↗