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

Myelin is a multilayered 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 signaling, with several implicated in neurological diseases. The cell surface abundance of the adhesion molecule neurofascin (NFASC), 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 NFASC 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 preferentially 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.

LipidQuant 1.0: automated data processing in lipid class separation-mass spectrometry quantitative workflows.

SUMMARY: We present the LipidQuant 1.0 tool for automated data processing workflows in lipidomic quantitation based on lipid class separation coupled with high-resolution mass spectrometry. Lipid class separation workflows, such as hydrophilic interaction liquid chromatography or supercritical fluid chromatography, should be preferred in lipidomic quantitation due to the coionization of lipid class internal standards with analytes from the same class. The individual steps in the LipidQuant workflow are explained, including lipid identification, quantitation, isotopic correction and reporting results. We show the application of LipidQuant data processing to a small cohort of human serum samples. AVAILABILITY AND IMPLEMENTATION: The LipidQuant 1.0 is freely available at Zenodo https://doi.org/10.5281/zenodo.5151201 and https://holcapek.upce.cz/lipidquant.php. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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

Reversed-phase ultrahigh-performance liquid chromatography-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-µm 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.

Simple and Reproducible Derivatization with Benzoyl Chloride: Improvement of Sensitivity for Multiple Lipid Classes in RP-UHPLC/MS.

The chemical derivatization of multiple lipid classes was developed using benzoyl chloride as a nonhazardous derivatization agent at ambient conditions. The derivatization procedure was optimized with standards for 4 nonpolar and 8 polar lipid classes and measured by reversed-phase ultrahigh-performance liquid chromatography-tandem mass spectrometry. The derivatization and nonderivatization approaches were compared on the basis of the calibration curves of 22 internal standards from 12 lipid classes. The new method decreased the limit of detection 9-fold for monoacylglycerols (0.9-1.0 nmol/mL), 6.5-fold for sphingoid base (0.2 nmol/mL), and 3-fold for diacylglycerols (0.9 nmol/mL). The sensitivity expressed by the ratio of calibration slopes was increased 2- to 10-fold for almost all investigated lipid classes and even more than 100-fold for monoacylglycerols. Moreover, the benzoylation reaction produces a more stable derivative of cholesterol in comparison to the easily in-source fragmented nonderivatized form and enabled the detection of fatty acids in a positive ion mode, which does not require polarity switching as for the nonderivatized form. The intralaboratory comparison with an additional operator without previous derivatization experiences shows the simplicity, robustness, and reproducibility. The stability of the derivatives was determined by periodical measurements during a one month period and five freeze/thaw cycles. The fully optimized derivatization method was applied to human plasma, which allows the detection of 169 lipid species from 11 lipid classes using the high confidence level of identification in reversed-phase (RP)-ultra high performance liquid chromatography (UHPLC)/mass spectrometry (MS). Generally, we detected more lipid species for monoacylglycerols, diacylglycerols, and sphingoid bases in comparison with previously reported papers without the derivatization.

Validation of lipidomic analysis of human plasma and serum by supercritical fluid chromatography-mass spectrometry and hydrophilic interaction liquid chromatography-mass spectrometry.

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) has a great potential for the high-throughput lipidomic quantitation of biological samples; therefore, the full optimization and method validation of UHPSFC/MS is compared here with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) in hydrophilic interaction liquid chromatography (HILIC) mode as the second powerful technique for the lipid class separation. First, the performance of six common extraction protocols is investigated, where the Folch procedure yields the best results with regard to recovery rate, matrix effect, and precision. Then, the full optimization and analytical validation for eight lipid classes using UHPSFC/MS and HILIC-UHPLC/MS methods are performed for the same sample set and applied for the lipidomic characterization of pooled samples of human plasma, human serum, and NIST SRM 1950 human plasma. The choice of appropriate internal standards (IS) for individual lipid classes has a key importance for reliable quantitative workflows illustrated by the selectivity while validation and the calculation of the quantitation error using multiple internal standards per lipid class. Validation results confirm the applicability of both methods, but UHPSFC/MS provides some distinct advantages, such as the successful separation of both non-polar and polar lipid classes unlike to HILIC-UHPLC/MS, shorter total run times (8 vs. 10.5 min), and slightly higher robustness. Various types of correlations between methods (UHPSFC/MS and HILIC-UHPLC/MS), biological material (plasma and serum), IS (laboratory and commercially mixtures), and literature data on the standard reference material show the intra- and inter-laboratory comparison in the quantitation of lipid species from eight lipid classes, the concentration differences in serum and plasma as well as the applicability of non-commercially available internal standard mixtures for lipid quantitation.

HILIC/MS quantitation of low-abundant phospholipids and sphingolipids in human plasma and serum: Dysregulation in pancreatic cancer.

A new hydrophilic interaction liquid chromatography - mass spectrometry method is developed for low-abundant phospholipids and sphingolipids in human plasma and serum. The optimized method involves the Cogent Silica type C hydride column, the simple sample preparation by protein precipitation, and the removal of highly abundant lipid classes using the postcolumn valve directed to waste during two elution windows. The method allows a highly confident and sensitive identification of low-abundant lipid classes in human plasma (246 lipid species from 24 lipid subclasses) based on mass accuracy and retention dependencies in both polarity modes. The method is validated for quantitation using two internal standards (if available) for each lipid class and applied to human plasma and serum samples obtained from patients with pancreatic ductal adenocarcinoma (PDAC), healthy controls, and NIST SRM 1950. Multivariate data analysis followed by various statistical projection methods is used to determine the most dysregulated lipids. Significant downregulation is observed for lysophospholipids with fatty acyl composition 16:0, 18:0, 18:1, and 18:2. Distinct trends are observed for phosphatidylethanolamines (PE) in relation to the bonding type of fatty acyls, where most PE with acyl bonds are upregulated, while ether/plasmenyl PE are downregulated. For the sphingolipid category, sphingolipids with very long N-acyl chains are downregulated, while sphingolipids with shorter N-acyl chains were upregulated in PDAC. These changes are consistently observed for various classes of sphingolipids, ranging from ceramides to glycosphingolipids, indicating a possible metabolic disorder in ceramide biosynthesis caused by PDAC.

Lipidomic profiling of human serum enables detection of pancreatic cancer.

Pancreatic cancer has the worst prognosis among all cancers. Cancer screening of body fluids may improve the survival time prognosis of patients, who are often diagnosed too late at an incurable stage. Several studies report the dysregulation of lipid metabolism in tumor cells, suggesting that changes in the blood lipidome may accompany tumor growth. Here we show that the comprehensive mass spectrometric determination of a wide range of serum lipids reveals statistically significant differences between pancreatic cancer patients and healthy controls, as visualized by multivariate data analysis. Three phases of biomarker discovery research (discovery, qualification, and verification) are applied for 830 samples in total, which shows the dysregulation of some very long chain sphingomyelins, ceramides, and (lyso)phosphatidylcholines. The sensitivity and specificity to diagnose pancreatic cancer are over 90%, which outperforms CA 19-9, especially at an early stage, and is comparable to established diagnostic imaging methods. Furthermore, selected lipid species indicate a potential as prognostic biomarkers.

Plasma lipidomic profiles of kidney, breast and prostate cancer patients differ from healthy controls.

Early detection of cancer is one of the unmet needs in clinical medicine. Peripheral blood analysis is a preferred method for efficient population screening, because blood collection is well embedded in clinical practice and minimally invasive for patients. Lipids are important biomolecules, and variations in lipid concentrations can reflect pathological disorders. Lipidomic profiling of human plasma by the coupling of ultrahigh-performance supercritical fluid chromatography and mass spectrometry is investigated with the aim to distinguish patients with breast, kidney, and prostate cancers from healthy controls. The mean sensitivity, specificity, and accuracy of the lipid profiling approach were 85%, 95%, and 92% for kidney cancer; 91%, 97%, and 94% for breast cancer; and 87%, 95%, and 92% for prostate cancer. No association of statistical models with tumor stage is observed. The statistically most significant lipid species for the differentiation of cancer types studied are CE 16:0, Cer 42:1, LPC 18:2, PC 36:2, PC 36:3, SM 32:1, and SM 41:1 These seven lipids represent a potential biomarker panel for kidney, breast, and prostate cancer screening, but a further verification step in a prospective study has to be performed to verify clinical utility.

Determination of one year stability of lipid plasma profile and comparison of blood collection tubes using UHPSFC/MS and HILIC-UHPLC/MS.

Effects of blood collection tubes, the time period, the sample origin, and the method used on the lipidomic profile are investigated by ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS) and hydrophilic interaction liquid chromatography ultrahigh-performance liquid chromatography - mass spectrometry (HILIC-UHPLC/MS). Heparin plasma samples have been obtained from 99 healthy volunteers at three time points separated by six-month intervals together with one collection for EDTA plasma and serum. Furthermore, lipid concentrations in heparin plasma collected at two different sites are compared. 171 lipid species from eight lipid classes are quantified with UHPSFC/MS, and 122 lipid species from four lipid classes with HILIC-UHPLC/MS. The accuracy of both methods is monitored by the quantitation error using two internal standards (IS) per individual lipid classes. No significant differences in lipid profiles are observed for different time points and types of collection tubes (heparin plasma, EDTA plasma, and serum). Most pronounced lipid concentration differences are observed for the comparison of NIST SRM 1950 human plasma and mean lipid concentrations of the investigated cohort. Furthermore, differences in lipid concentrations are observed between employed methods (UHPSFC/MS vs. HILIC-UHPLC/MS), which can be compensated by the normalization using NIST SRM 1950 human plasma used as the quality control sample.

Lipidomic characterization of exosomes isolated from human plasma using various mass spectrometry techniques.

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 µm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in <7 minute run time. MALDI provided the complementary information on anionic lipid classes, such as sulfatides. The nontargeted analysis of 12 healthy volunteers was performed, and absolute molar concentration of 244 lipids in exosomes and 191 lipids in plasma belonging to 10 lipid classes were quantified. The statistical evaluation of data included principal component analysis, orthogonal partial least square discriminant analysis, S-plots, p-values, T-values, fold changes, false discovery rate, box plots, and correlation plots, which resulted in the information on lipid changes in exosomes in comparison to plasma. The major changes were detected in the composition of triacylglycerols, diacylglycerols, phosphatidylcholines, and lysophosphatidylcholines, whereby sphingomyelins, phosphatidylinositols, and sulfatides showed rather similar profiles in both biological matrices.

Reversed-Phase Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Method for High-Throughput Lipidomic Quantitation.

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method is optimized for the quantitation of a large number of lipid species in biological samples, primarily in human plasma and serum. The method uses a C18 bridged ethylene hybrid (BEH) column (150 × 2.1 mm; 1.7 µm) for the separation of lipids from 23 subclasses with a total run time of 25 min. Lipid species separation allows the resolution of isobaric and isomeric lipid forms. A triple quadrupole mass spectrometer is used for targeted lipidomic analysis using multiple reaction monitoring (MRM) in the positive ion mode. Data are evaluated by Skyline software, and the concentrations of analytes are determined using internal standards per each individual lipid class.

Ultrahigh-Performance Supercritical Fluid Chromatography-Mass Spectrometry in the Clinical Lipidomic Analysis.

Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) method is optimized for the high-throughput quantitation of lipids in human serum and plasma with an emphasis on robustness and accurate quantitation. Bridged ethylene hybrid (BEH) silica column (100 × 3 mm; 1.7 µm) is used for the separation of 17 nonpolar and polar lipid classes in 4.4 min using the positive ion electrospray ionization mode. The lipid class separation approach in UHPSFC/MS results in the coelution of all lipid species within one lipid class in one chromatographic peak, including two exogenous internal standards (IS) per lipid class, which provides the optimal conditions for robust quantitation. The method was validated according to European Medicines Agency and Food and Drug Administration recommendations. UHPSFC/MS combined with LipidQuant software allows a semiautomated process to determine lipid concentrations with a total run time of only 8 min including column equilibration, which enables the analysis of 160 samples per day.