Combining Mass Spectrometry with Paterno-Buchi Reaction to Determine Double-Bond Positions in Lipids at the Single-Cell Level.
ABSTRACT: Single cell MS (SCMS) techniques are under rapid development for molecular analysis of individual cells among heterogeneous populations. Lipids are basic cellular constituents playing essential functions in energy storage and the cellular signaling processes of cells. Unsaturated lipids are characterized with one or multiple carbon-carbon double (C?C) bonds, and they are critical for cell functions and human diseases. Characterizing unsaturated lipids in single cells allows for better understanding of metabolomic biomarkers and therapeutic targets of rare cells (e.g., cancer stem cells); however, these studies remain challenging. We developed a new technique using a micropipette needle, in which Paternò-Büchi (PB) reactions at C?C bond can be induced, to determine locations of C?C bonds in unsaturated lipids at the single-cell level. The micropipette needle is produced by combining a pulled glass capillary needle with a fused silica capillary. Cell lysis solvent and PB reagent (acetone or benzophenone) are delivered into the micropipette needle (tip size ? 15 um) through a fused silica capillary. The capillary needle plays multiple functions (i.e., single cell sampling probe, cell lysis container, microreactor, and nano-ESI emitter) in the experiments. Both regular (no reaction) and reactive (with PB reaction) SCMS analyses of the same cell can be achieved. C?C bond locations were determined from MS scan and MS/MS of PB products assisted by Python programs. This technique can potentially be used for other reactive SCMS studies to enhance molecular analysis for broad ranges of single cells.
Project description:Existing single cell mass spectrometry (SCMS) sampling platforms are largely designed to work only with immobilized cells and not the suspended cells isolated from patient samples. Here, we present a novel method that integrates a commercially available cell manipulation system commonly used for in vitro fertilization with the Single-probe SCMS sampling technology. The combined Single-probe SCMS/cell manipulating platform is capable of rapidly analyzing intracellular species in real time from a suspension leukemia cell line. A broad range of molecular species was detected, and species of interest were verified using tandem MS (MS/MS). Experimental results were analyzed utilizing statistical analyses such as principle component analysis (PCA) and t-tests. The developed SCMS/cell manipulation system is a versatile tool to provide rapid single cell analysis of broad types of patient cell samples.
Project description:The exploration of single cells reveals cell heterogeneity and biological principle of cellular metabolism. Although a number of mass spectrometry (MS) based single cell MS (SCMS) techniques have been dedicatedly developed with high efficiency and sensitivity, limitations still exist. In this work, we introduced a microscale multifunctional device, the T-probe, which integrates cellular contents extraction and immediate ionization, to implement online in situ SCMS analysis at ambient conditions with minimal sample preparation. With high sensitivity and reproducibility, the T-probe was employed for MS analysis of single HeLa cells under control and anticancer drug treatment conditions. Intracellular species and xenobiotic metabolites were detected, and changes of cellular metabolic profiles induced by drug treatment were measured. Combining SCMS experiments with statistical data analyses, including Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) and two-sample t-test, we provided biological insights into cellular metabolic response to drug treatment. Online MS/MS analysis was conducted at single cell level to identify species of interest, including endogenous metabolites and the drug compound. Using the T-probe SCMS technique combined with comprehensive data analyses, we provide an approach to understanding cellular metabolism and evaluate chemotherapies at the single cell level.
Project description:We conducted single cell metabolomics studies of live cancer cells through online single cell mass spectrometry (SCMS) experiments combined with a generalized comprehensive data analysis workflow. The SCMS experiments were carried out using the Single-probe device coupled with a mass spectrometer to measure molecular profiles of cells in response to two mitotic inhibitors, taxol and vinblastine, under a series of treatment conditions. SCMS metabolomic data were analyzed using a comprehensive approach, including data pre-treatment, visualization, statistical analysis, machine learning, and pathway enrichment analysis. For comparative studies, traditional liquid chromatography-MS (LC-MS) experiments were conducted using lysates prepared from bulk cell samples. Metabolomic profiles of single cells were visualized through Partial Least Square-Discriminant Analysis (PLS-DA), and the phenotypic biomarkers associated with emerging phenotypes induced by drug treatment were discovered and compared through a series of rigorous statistical analysis. Species of interest were further identified at both the single cell and population levels. In addition, four biological pathways potentially involved in the drug treatment were determined through pathway enrichment analysis. Our work demonstrated the capability of comprehensive pipeline studies of single cell metabolomics. This method can be potentially applied to broader types of SCMS datasets for future pharmaceutical and chemotherapeutic research.
Project description:Techniques that allow single cell analysis are gaining widespread attention, and most of these studies utilize genomics-based approaches. While nanofluidic technologies have enabled mass spectrometric analysis of single cells, these measurements have been limited to metabolomics and lipidomic studies. Single cell proteomics has the potential to improve our understanding of intercellular heterogeneity. However, this approach has faced challenges including limited sample availability, as well as a requirement of highly sensitive methods for sample collection, cleanup, and detection. We present a technique to overcome these limitations by combining a micropipette (pulled glass capillary) based sample collection strategy with offline sample preparation and nanoLC-MS/MS to analyze proteins through a bottom-up proteomic strategy. This study explores two types of proteomics data acquisition strategies namely data-dependent (DDA) and data-independent acquisition (DIA). Results from the study indicate DIA to be more sensitive enabling analysis of >1600 proteins from ?130 ?m Xenopus laevis embryonic cells containing <6 nL of cytoplasm. The method was found to be robust in obtaining reproducible protein quantifications from single cells spanning the 1-128-cell stages of development. Furthermore, we used micropipette sampling to study intercellular heterogeneity within cells in a single embryo and investigated embryonic asymmetry along both animal-vegetal and dorsal-ventral axes during early stages of development. Investigation of the animal-vegetal axis led to discovery of various asymmetrically distributed proteins along the animal-vegetal axis. We have further compared the hits found from our proteomic data sets with other studies and validated a few hits using an orthogonal imaging technique. This study forms the first report of vegetal enrichment of the germ plasm associated protein DDX4/VASA in Xenopus embyos. Overall, the method and data presented here holds promise to enable important leads in developmental biology.
Project description:Mass spectrometry analysis of cholesteryl esters (CEs) faces several challenges, with one of them being the determination of the carbon-carbon double bond (C=C) locations within unsaturated fatty acyl chains. Patern?-Büchi (PB) reaction, a photochemical reaction based on the addition of acetone to C=C, is capable of C=C location determination when coupled with tandem mass spectrometry (MS/MS). In this study, the PB reaction conditions were tailored for CEs and subsequent nanoelectrospray ionization (nanoESI). A solvent system containing acetone/methanol/dichloromethane/water (40/30/20/10, volume ratios) and 100 ?M LiOH was determined to be optimal, resulting in reasonable PB reaction yield (~30%) and good ionization efficiency (forming lithium adduct of CEs). Collision-induced dissociation (CID) of the PB reaction products produced characteristic fragment ions of CE together with those modified by the PB reactions, such as lithiated fatty acyl ([FA + Li]+) and its PB product ([FA - PB + Li]+). MS3 CID of [FA - PB + Li]+ led to abundant C=C diagnostic ion formation, which was used for C=C location determination and isomer quantitation. A PB-MS3 CID approach was developed and applied for CE analysis from human plasma. A series of unsaturated CEs was identified with specific C=C locations within fatty acyl chains. Absolute quantitation for each CE species was achieved including coexisting C=C location isomers, such as ?9 and ?11 isomers of CE 18:1 and ?-6 and ?-3 isomers of CE 18:3. These results show that PB-MS/MS is useful in uncovering structural diversity of CEs due to unsaturation in fatty acyls, which is often undetected from current lipid analysis approach. Graphical Abstract ?.
Project description:Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.
Project description:Lipid dysregulation has been implicated in multiple sclerosis due to its involvement during and after inflammation. In this study, we have profiled fatty acids (FAs) in the mouse model of multiple sclerosis with new capabilities of assigning carbon-carbon double bond (C=C) location(s) and quantifying C=C location isomers. These new capabilities are enabled by pairing the solution phase Paternò-Büchi (PB) reaction that modifies C=C bonds in FAs, with tandem mass spectrometry (MS/MS), termed as PB-MS/MS. A series of unsaturated FAs and C=C location isomers have been identified, including FA17:1 (?10), FA18:1 (?9 and ?11), FA18:2 (?9 and ?12), and FA 20:4 (?5, ?8, ?11, ?14). Notable differences in saturated and unsaturated FAs between normal and experimental autoimmune encephalomyelitis (EAE) mice spinal cords have been detected. Furthermore, the effects of hydralazine, a scavenger of acrolein, on profile changes of FAs in mice were studied. Increased ?11-to-?9 isomer ratios for FA 18:1 were noted in the diseased samples as compared to the control. The present work provides a facile and robust analytical method for the quantitation of unsaturated FAs as well as identification of FA C=C location isomers, which will facilitate discovering prospective lipid markers in multiple sclerosis.
Project description:Cancer stem cells (CSCs) are rare types of cells responsible for tumor development, relapse, and metastasis. However, current research in CSC biology is largely limited by the difficulty of obtaining sufficient CSCs. Single-cell analysis techniques are promising tools for CSC-related studies. Here, we used the Single-probe mass spectrometry (MS) technique to investigate the metabolic features of live colorectal CSCs at the single-cell level. Experimental data were analyzed using statistical analysis methods, including the t-test and partial least squares discriminant analysis. Our results indicate that the overall metabolic profiles of CSCs are distinct from non-stem cancer cells (NSCCs). Specifically, we demonstrated that tricarboxylic acid (TCA) cycle metabolites are more abundant in CSCs compared to NSCCs, indicating their major energy production pathways are different. Moreover, CSCs have relatively higher levels of unsaturated lipids. Inhibiting the activities of stearoyl-CoA desaturase-1 (SCD1), nuclear factor ?B (NF-?B), and aldehyde dehydrogenases (ALDH1A1) in CSCs significantly reduced the abundances of unsaturated lipids and hindered the formation of spheroids, resulting in reduced stemness of CSCs. Our techniques and experimental protocols can be potentially used for metabolomic studies of other CSCs and rare types of cells and provide a new approach to discovering functional biomarkers as therapeutic targets.
Project description:Lipidomics analysis for large-scale studies aiming at the identification and quantification of natural lipidomes is often performed using LC-MS-based data acquisition. However, the choice of suitable LC-MS method for accurate lipid quantification remains a matter of debate. Here, we performed the systematic comparison between two HRAM-MS-based quantification workflows based on HILIC and RPLC MS by quantifying 191 lipids from five lipid classes in human blood plasma using deuterated standards in the "one ISTD-per-lipid class" approach. Lipid quantification was performed considering all necessary isotopic corrections, and obtained correction factors are illustrated. Concentrations of lipids in NIST® SRM® 1950 human blood plasma determined by the two methods were comparable for most of the studied lipid species except for highly unsaturated phosphatidylcholines (PC). A comparison of lipid concentrations to consensus values determined in a previously published multi-laboratory study illustrated possible "overestimation" of concentrations for these highly unsaturated lipids by HILIC MS. We evaluated the influence of lipid loading amounts as well as the difference between quantified lipid and internal standard concentrations on the HILIC MS quantification results. We conclude that both HILIC and RPLC HRAM-MS workflows can be equally used for accurate lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM) lipid quantification, despite significant differences in the concentration of highly unsaturated PC lipids which need to be addressed by establishing response factors to account for the differences in degree of lipid unsaturation. Graphical.
Project description:Lipids are highly diverse biomolecules associated with several biological functions including structural constituent, energy storage, and signal transduction. It is essential to characterize lipid structural isomers and further understand their biological roles. Unsaturated lipids contain one or multiple carbon-carbon double bonds. Identifying double bond position presents a major challenge in unsaturated lipid characterization. Recently, several advancements have been made for double bond localization by mass spectrometry (MS) analysis. However, many of these studies require complex chemical reactions or advanced mass spectrometers with special fragmentation techniques, which limits the application in lipidomics study. Here, an innovative meta-chloroperoxybenzoic acid ( m-CPBA) epoxidation reaction coupling with collision-induced dissociation (CID)-MS/MS strategy provides a new tool for unsaturated lipidomics analysis. The rapid epoxidation reaction was carried out by m-CPBA with high specificity. Complete derivatization was achieved in minutes without overoxidized byproduct. Moreover, diagnostic ion pair with 16 Da mass difference indicated localization of carbon-carbon double bond in MS/MS spectra. Multiple lipid classes were evaluated with this strategy and generated abundant fragments for structural analysis. Unsaturated lipid analysis of yeast extract using this strategy took less than 30 min, demonstrating the potential for high-throughput lipidomics analysis by this approach. This study opens a door for high throughput unsaturated lipid analysis with minimal requirement for instrumentation, which could be widely applied in lipidomics analysis.