Characterization and Quantification of Diacylglycerol Species in Biological Extracts after One-Step Derivatization: A Shotgun Lipidomics Approach
ABSTRACT Diacylglycerols (DAGs) are important intermediates of lipid metabolism and cellular signaling. It is well known that the mass levels of DAG are altered under disease states. Therefore, quantitative analysis of DAGs in biological samples can provide critical information to uncover underlying mechanisms of various cellular functional disorders. Although great efforts on the analysis of individual DAG species have recently been made by utilizing mass spectrometry with or without derivatization, cost effective and high throughput methodology for identification and quantification of all DAG species including regioisomers, particularly in an approach of shotgun lipidomics, are still missing. Herein, we described a novel method for directly identifying and quantifying DAG species including regioisomers present in lipid extracts of biological samples after facile one-step derivatization with dimethylglycine based on the principles of multi-dimensional mass spectrometry-based shotgun lipidomics. The established method provided substantial sensitivity (low limit of quantification at amol/μl), high specificity, and broad linear dynamics range (2,500 folds) without matrix effects. By exploiting this novel method, we revealed a 16-fold increase of total DAG mass in the livers of ob/ob mice compared to their wild type controls at 4 months of age (an insulin-resistant state) vs. a 5-fold difference between 3-month old mice (with normal insulin). These results demonstrated the importance and power of the method for studying biochemical mechanisms underpinning disease states.
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ABSTRACT: Lipidomics, a branch of metabolomics, is the large-scale study of pathways and networks of all cellular lipids in biological systems such as cells, tissues or organisms. The recent advance in mass spectrometry technologies have enabled more comprehensive lipid profiling in the biological samples. In this review, we compared four representative lipid profiling technoligies including GC-MS, LC-MS, direct infusion-MS and imaging-MS. We also summarized representative lipid database, and further discussed the applications of lipidomics to the diagnostics of various diseases such as diabetes, obesity, hypertension, and Alzheimer diseases.03/2014; 4(1):17-33. DOI:10.15280/jlm.2014.4.1.17
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ABSTRACT: The emerging field of sphingolipidomics calls for accurate quantitative analyses of sphingolipidome. Existing analytical methods for sphingolipid (SPL) profiling often suffer from isotopic/isomeric interference, leading to the low-abundant, but biologically important SPLs being undetected. In the current study, we have developed an improved sphingolipidomic approach for reliable and sensitive quantification of up to 10 subclasses of cellular SPLs. By integratively utilizing high efficiency chromatographic separation, quadrupole time of flight (Q-TOF) and triple quadrupole (QQQ) mass spectrometry (MS), our approach facilitated unambiguous identification of several groups of potentially important but low-abundant SPLs that are usually masked by isotopic/isomeric species and hence largely overlooked in many published methods. The methodology, which featured a modified sample preparation and optimized MS parameters, permitted the measurement of 86 individual SPLs in PC12 cells in a single run, demonstrating great potential for high throughput analysis. The improved characterization, along with increased sensitivity for low-abundant SPL species, resulted in the highest number of SPLs being quantified in literature to date. The improved method was fully validated and applied to a lipidomic study of PC 12 cell samples with or without amyloid β peptide (Aβ) treatment, which presents a most precise and genuine sphingolipidomic profile of the PC12 cell line. The adoption of the metabolomic protocol, as described in this study, could avoid misidentification and bias in the measurement of the analytically-challenging low-abundant endogenous SPLs, hence achieving informative and reliable sphingolipidomics data relevant to discovery of potential SPL biomarkers for Aβ-induced neurotoxicity and neurodegenerative disease.Analytical Chemistry 05/2014; 86(12). DOI:10.1021/ac5009964 · 5.83 Impact Factor
Article: Trimethylation Enhancement Using Diazomethane (TrEnDi) II: Rapid In-Solution Concomitant Quaternization of Glycerophospholipid Amino Groups and Methylation of Phosphate Groups via Reaction with Diazomethane Significantly Enhances Sensitivity in Mass Spectrometry Analyses via a Fixed, Permanent Positive Charge[Show abstract] [Hide abstract]
ABSTRACT: A novel mass spectrometry (MS)-based lipidomics strategy that exposes glycerophospholipids to an ethereal solution of diazomethane and acid, derivatizing them to contain a net fixed, permanent positive charge is described. The sensitivity of modified lipids to MS detection is enhanced via improved ionization characteristics as well as consolidation of ion dissociation to form one or two strong, characteristic polar head group fragments. Our strategy has been optimized to enable a priori prediction of ion fragmentation patterns for four subclasses of modified glycerophospholipid species. Our method enables analyte ionization regardless of proton affinity, thereby decreasing ion suppression and permitting predictable precursor ion-based quantitation with improved sensitivity in comparison to MS-based methods that are currently used on unmodified lipid precursors.Analytical Chemistry 09/2014; 86(19). DOI:10.1021/ac501588y · 5.83 Impact Factor