Lipid profiling of lipoproteins by electrospray ionization tandem mass spectrometry

Lipoproteins are of fundamental importance for the lipid transport and cardiovascular disease. The function and metabolism of lipoproteins is intimately linked to the biophysical properties of their surface lipids. Although a number of disease associations were found for lipid species in plasma, only a few studies reported lipid profiles of lipoproteins. Here, we provide an overview of techniques for lipoprotein separation, methods for lipid species analysis based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) as well as data from recent lipidomic studies on lipoprotein fractions. We also discuss the different analytical strategies and how lipid profiling can expand our understanding of the biology and structures of lipoproteins.     

Sphingolipid profiling of human plasma and FPLC-separated lipoprotein fractions by hydrophilic interaction chromatography tandem mass spectrometry

Sphingolipids comprise bioactive molecules which are known to play important roles both as intracellular and extracellular signalling molecules. Here we used a previously developed hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS/MS) method to profile plasma sphingolipids. Method validation showed sufficient precision and sensitivity for application in large clinical studies. Sample stability testing demonstrated that immediate plasma separation is important to achieve reliable results. Analysis of plasma from 25 healthy blood donors revealed a comprehensive overview of free sphingoid base, sphingosylphosphorylcholine (SPC), hexosylceramide (HexCer), lactosylceramide (LacCer), and ceramide-1-phosphate (Cer1P) species level. Besides the major sphingoid base sphingosine (d18:1), we found d16:1 and d18:2 species in most of these lipid classes. Interestingly, pronounced differences were detected in the species profiles of HexCer and LacCer. Additionally, sphingolipids were quantified in lipoprotein fractions prepared by fast performance liquid chromatography (FPLC). HexCer and LacCer showed similar distributions with about 50% in LDL, 40% in HDL and less than 10% in the VLDL fraction. More than 90% of sphingoid base phosphates were found in HDL and albumin containing fractions. In summary, HILIC-MS/MS provides a valuable tool to profile minor sphingolipid species in plasma and in lipoprotein fractions. Comparing profiles from tissues or blood cells, these species profiles may help to address the origin of plasma sphingolipids.     

Identification and characterization by electrospray mass spectrometry of endogenous Drosophila sphingadienes

Sphingolipids comprise a complex group of lipids concentrated in membrane rafts and whose metabolites function as signaling molecules. Sphingolipids are conserved in Drosophila, in which their tight regulation is required for proper development and tissue integrity. In this study, we identified a new family of Drosophila sphingolipids containing two double bonds in the long chain base (LCB). The lipids were found at low levels in wild-type flies and accumulated markedly in Drosophila Sply mutants, which do not express sphingosine-1-phosphate lyase and are defective in sphingolipid catabolism. To determine the identity of the unknown lipids, purified whole fly lipid extracts were separated on a C18-HPLC column and analyzed using electrospray mass spectrometry. The lipids contain a LCB of either 14 or 16 carbons with conjugated double bonds at C4,6. The Delta(4,6)-sphingadienes were found as free LCBs, as phosphorylated LCBs, and as the sphingoid base in ceramides. The temporal and spatial accumulation of Delta(4,6)-sphingadienes in Sply mutants suggests that these lipids may contribute to the muscle degeneration observed in these flies.     

Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry

Neutral lipids are an important class of hydrophobic compounds found in all cells that play critical roles from energy storage to signal transduction. Several distinct structural families make up this class, and within each family there are numbers of individual molecular species. A solvent extraction protocol has been developed to efficiently isolate neutral lipids without complete extraction of more polar phospholipids. Normal-phase HPLC was used for the separation of cholesteryl esters (CEs), monoalkylether diacylglycerols, triacylglycerols, and diacylglycerols in a single HPLC run from this extract. Furthermore, minor lipids such as ubiquinone-9 could be detected in RAW 264.7 cells. Molecular species that make up each neutral lipid class can be analyzed both qualitatively and quantitatively by on-line LC-MS and LC-MS/MS strategies. The quantitation of >20 CE molecular species revealed that challenging RAW 264.7 cells with a Toll-like receptor 4 agonist caused a >20-fold increase in the content of CEs within cells, particularly those CE molecular species that contained saturated (14:0, 16:0, and 18:1) fatty acyl groups. Longer chain CE molecular species did not change in response to the activation of these cells.     

Monoacylglycerol accumulation in low and high density lipoproteins during the hydrolysis of very low density lipoprotein triacylglycerol by lipoprotein lipase.

We have demonstrated that low and high density lipoproteins from monkey plasma are capable of accepting and accumulating monoacylglycerol that is formed by the action of lipoprotein lipase on monkey lymph very low density lipoproteins. Furthermore, the monoacylglycerol that accumulates in both low and high density lipoproteins is not susceptible to further hydrolysis by lipoprotein lipase but is readily degraded by the monoacylglycerol acyltransferase of monkey liver plasma membranes. These observations suggest a new mechanism for monoacylglycerol transfer from triacylglycerol rich lipoproteins to other lipoproteins. In addition, the finding that monoacylglycerol bound to low and high density lipoprotein is degraded by the liver enzyme but not lipoprotein lipase lends support to the hypothesis that there are distinct and consecutive extrahepatic and hepatic stages in the metabolism of triacylglycerol in plasma lipoproteins.     

Poppy alkaloid profiling by electrospray tandem mass spectrometry and electrospray FT-ICR mass spectrometry after [ring-13C6]-tyramine feeding

Papaver alkaloids play a major role in medicine and pharmacy. In this study, [ring-(13)C(6)]-tyramine as a biogenetic precursor of these alkaloids was fed to Papaver somniferum seedlings. The alkaloid pattern was elucidated both by direct infusion high-resolution ESI-FT-ICR mass spectrometry and liquid chromatography/electrospray tandem mass spectrometry. Thus, based on this procedure, the structure of about 20 alkaloids displaying an incorporation of the labeled tyramine could be elucidated. These alkaloids belong to different classes, e.g. morphinan, benzylisoquinoline, protoberberine, benzo[c]phenanthridine, phthalide isoquinoline and protopine. The valuable information gained from the alkaloid profile demonstrates that the combination of these two spectrometric methods represents a powerful tool for evaluating biochemical pathways and facilitates the study of the flux of distant precursors into these natural products.     

Characterization of a noncovalent lipocalin complex by liquid chromatography/electrospray ionization mass spectrometry.

Nanoscale liquid chromatography coupled to electrospray ionization mass spectrometry was used to identify the nature of the ligand that binds noncovalently to siderocalin (lipocalin 2). The folded state siderocalin-ligand complex was separated from free, unfolded siderocalin using reversed phase chromatography, and the molecular weight of the siderocalin ligand was then determined from the deconvoluted molecular weights of the complex and of the free protein. The ligand was identified as dihydroxybenzoyl-serine, a breakdown product of enterobactin, an iron-chelating compound ("siderophore") synthesized in bacteria. These results demonstrate that, in some cases, electrostatic noncovalent protein complexes can survive the denaturing conditions of reversed phase liquid chromatography and the gas phase transfer occurring during electrospray ionization.     

Proteomic profiling of erythrocyte proteins by proteolytic digestion chip and identification using two-dimensional electrospray ionization tandem mass spectrometry

Self-assembled monolayers (SAMs) on coinage metal provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition, and other interfacial phenomena. The bonding of enzyme to SAMs of alkanethiols onto gold surfaces is exploited to produce an enzyme chip. In this work, the attachment of trypsin to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water soluble N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as coupling agent. A two-dimensional liquid-phase separation scheme coupled with mass spectrometry is presented for proteomic analysis of erythrocyte proteins. The application of proteomics, particularly with reference to analysis of proteins, will be described. Surface analyses have revealed that the X-ray Photoelectron Spectroscopy (XPS) C1s and N1s core levels illustrate the immobilization of trypsin. These data are also in good agreement with Fourier Transformed Infrared Reflection-Attenuated Total Reflection (FTIR-ATR) spectra for the peaks at Amide I and Amide II. Using two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system observations, analytical results have demonstrated the erythrocyte proteins digestion of the immobilized trypsin on the functionalized SAMs surface. For such surfaces, it also shows the enzyme digestion ability of the immobilized trypsin. The experiment results revealed the identification of 272 proteins from erythrocyte protein sample. The terminal groups of the SAMs structure can be further functionalized with biomolecules or antibodies to develop surface-base diagnostics, biosensors, or biomaterials.     

Determination of iodide in urine using electrospray ionization tandem mass spectrometry

A rapid and sensitive electrospray ionization (ESI) tandem mass spectrometry (MS–MS) procedure was developed for the determination of iodide (I⁻). A gold (Au) and I⁻ complex was formed immediately after the addition of the chelating agent NaAuCl₄ to I⁻ solution, and was extracted with methyl isobutyl ketone. One to five microliters of the extract were injected directly into an ESI–MS–MS instrument. I⁻ quantification was performed by selecting reaction monitoring of the product ion I⁻ at m/z 127 derived from the precursor ion ¹⁹⁷AuI₂⁻ at m/z 451. I⁻ concentration was measured in the quantification range from 10⁻⁷ to 10⁻⁵ M using 50 μL of solution within 10 min. Iodate was reduced to I⁻ with ascorbic acid and determined. I⁻ concentration in reference urine 2670a was measured after treatments.     

Focused lipidomics by tandem mass spectrometry

In this paper we performed focused analyses of phospholipids by using the data of precursor ion scanning and neutral loss scanning of their polar head groups and fatty acyl moieties for the specific search of categorical phospholipids. By using precursor ion scanning or neutral loss scanning of polar head groups in the positive ion mode, more sensitive identification were obtained than that in the negative ion mode. Precursor ion scanning of carbonic anions in the negative ion mode was also effective to identify molecular species of phospholipids having specified fatty acyl moieties. By using these analytical methods, the detection limits of individual metabolites are going up to 5-20-fold of former conventional methods. The important factor is that by focusing in some limited categories of molecules, detection limit is greatly enhanced, thus minor but important molecules can be detected. Moreover, combination of LC-MS/MS and focused scanning for head group was revealed to be useful to identify very minor molecular species in the focused class of phospholipids.     

Glycan profiling of monoclonal antibodies using zwitterionic-type hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry detection

We present a new method for the analysis of glycans enzymatically released from monoclonal antibodies (MAbs) employing a zwitterionic-type hydrophilic interaction chromatography (ZIC–HILIC) column coupled with electrospray ionization mass spectrometry (ESI–MS). Both native and reduced glycans were analyzed, and the developed procedure was compared with a standard HILIC procedure used in the pharmaceutical industry whereby fluorescent-labeled glycans are analyzed using a TSK Amide-80 column coupled with fluorescence detection. The separation of isobaric alditol oligosaccharides present in monoclonal antibodies and ribonuclease B is demonstrated, and ZIC–HILIC is shown to have good capability for structural recognition. Glycan profiles obtained with the ZIC–HILIC column and ESI–MS provided detailed information on MAb glycosylation, including identification of some less abundant glycan species, and are consistent with the profiles generated with the standard procedure. This new ZIC–HILIC method offers a simpler and faster approach for glycosylation analysis of therapeutic antibodies.     

High-sensitivity determination of tyrosine-phosphorylated peptides by on-line enzyme reactor and electrospray ionization mass spectrometry.

We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on-line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase beta onto the inner surface of a small piece of a 50-microns inner diameter, 360-microns outer diameter fused silica capillary or by immobilization of the phosphatase onto 40-90-microns avidin-activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on-line electrospray ionization mass spectrometer. Tyrosine-phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine-phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied.     

Determination of metolazone in human blood by liquid chromatography with electrospray ionization tandem mass spectrometry

A rapid, sensitive and accurate liquid chromatographic-tandem mass spectrometric method is described for the determination of metolazone in human blood. Metolazone was extracted from blood using ethyl acetate and separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase consisting of a mixture of acetonitrile, 10 mmol/l ammonium acetate and formic acid (60:40:0.1, v/v/v) was delivered at a flow rate of 0.5 ml/min. Electrospray ionization (ESI) source was operated in positive ion mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 366-->m/z 259 and m/z 321-->m/z 275 were used to quantify metolazone and the lorazepam (internal standard), respectively. The linearity was obtained over the concentration range of 0.5-500 ng/ml for metolazone and the lower limit of quantitation (LLOQ) was 0.5 ng/ml. For each level of QC samples, inter- and intra-run precision was less than 8.07 and 3.56% (relative standard deviation (RSD)), respectively, and the bias was within +/-4.0%. This method was successfully applied to the pharmacokinetic study of metolazone formulation after oral administration to humans.     

Identification of oxidized phospholipids by electrospray ionization mass spectrometry and LC-MS using a QQLIT instrument

Phospholipids are complex and varied biomolecules that are susceptible to lipid peroxidation after attack by free radicals or electrophilic oxidants and can yield a large number of different oxidation products. There are many available methods for detecting phospholipid oxidation products, but also various limitations and problems. Electrospray ionization mass spectrometry allows the simultaneous but specific analysis of multiple species with good sensitivity and has a further advantage that it can be coupled to liquid chromatography for separation of oxidation products. Here, we explain the principles of oxidized phospholipid analysis by electrospray mass spectrometry and describe fragmentation routines for surveying the structural properties of the analytes, in particular precursor ion and neutral loss scanning. These allow targeted detection of phospholipid headgroups and identification of phospholipids containing hydroperoxides and chlorine, as well as the detection of some individual oxidation products by their specific fragmentation patterns. We describe instrument protocols for carrying out these survey routines on a QTrap5500 mass spectrometer and also for interfacing with reverse-phase liquid chromatography. The article highlights critical aspects of the analysis as well as some limitations of the methodology.     

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50 ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.     

Multidimensional profiling of plasma lipoproteins by size exclusion chromatography followed by reverse-phase protein arrays

The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 μl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-β-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique.     

Characterization of mycolic acids from the pathogen Rhodococcus equi by tandem mass spectrometry with electrospray ionization

We describe a simple tandem mass spectrometric approach toward structural characterization of mycolic acids, the long-chain α-alkyl-β-hydroxy fatty acids unique to mycobacteria and related taxa. On collisionally activated dissociation in a linear ion trap or tandem quadrupole mass spectrometer, the [M−H]⁻ ions of mycolic acid generated by electrospray ionization undergo dissociation to eliminate the meroaldehyde residue, leading to formation of carboxylate anions containing α-alkyl chains. The structural information from these fragment ions affords structural assignment of the mycolic acids, including the lengths of the meromycolate chain and the α-branch. This study revealed that the mycolic acids isolated from pathogenic Rhodococcus equi 103 contained a series of homologous ions having C₃₀ to C₅₀ chain with 0–2 double bonds. The α-branch ranged from C₁₀ to C₁₈ with 0 to 1 double bond, in which 16:0 and 14:0 are the most prominent, whereas the meromycolate chain ranged from C₁₄ to C₃₄ with 0 to 2 double bonds. The major molecular species consisted of more than 3 isomers that differ by the lengths of the α-branch or meromycolate chain, and up to 10 isobaric isomers were identified for some minor ions. We also employed tandem quadrupole mass spectrometry with precursor ion and neutral loss scans for profiling mycolic acid with specific structure in mixtures. The tandem spectra obtained from precursor ion scans of m/z 255 (16:0-carboxylate anion) and m/z 227 (14:0-carboxylate anion) may provide a simple specific means for classification of Rhodococci species, whereas tandem spectra from neutral loss of meroaldehyde residue scans provided a simple approach to reveal the mycolic acid molecules with specific meromycolate chain in mixtures.     

Simultaneous measurement of tryptophan and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry

We have expanded a liquid chromatographic-tandem mass spectrometric method that measures 3-hydroxykynurenine and 3-hydroxyanthranilic acid in addition to tryptophan and kynurenine both intra- and extracellularly. After reversed phase HPLC separation, the compounds were detected in the MS positive multiple reaction monitoring mode. We found a good linear response for each tryptophan metabolite. The lower limit of quantification for each compound ranged from 0.01 to 0.1 microM. The extraction efficiencies from spiked cell samples and culture medium ranged between 83 and 111% and the overall coefficient of variation of analyses was less than 7%. Using our method, we found tryptophan metabolites in the cells and the culture medium of LN229 human glioma cells were stimulated by interferon-gamma, a known inducer of indoleamine 2,3-dioxygenase. The intracellular concentrations of kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid were higher than those in the medium. This is the first report of a method for the simultaneous determination of tryptophan and its metabolic products both intra- and extracellularly.     

Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry

Sphingosine (SPH) comprises the backbone of sphingolipids and is known as a second messenger involved in the modulation of cell growth, differentiation, and apoptosis. The currently available methods for the quantification of SPH are, in part, complicated, time-consuming, insensitive, or unselective. Therefore, a fast and convenient methodology for the quantification of SPH and the biosynthetic intermediate sphinganine (SPA) was developed. The method is based on an HPLC separation coupled to electrospray ionization tandem mass spectrometry (MS/MS). Quantitation is achieved by the use of a constant concentration of a non-naturally occurring internal standard, 17-carbon chain SPH (C17-SPH), together with a calibration curve established by spiking different concentrations of naturally occurring sphingoid bases. SPH and SPA coeluted with C17-SPH, which allows an accurate correction of the analyte response. Interference of the SPH+2 isotope with SPA quantification was corrected by an experimentally determined factor. The limits of detection were 9 fmol for SPH and 21 fmol for SPA. The overall coefficients of variation were 8% and 13% for SPH and SPA, respectively. The developed HPLC-tandem mass spectrometry methodology, with an analysis time of 3.5 min, simple sample preparation, and automated data analysis, allows high-throughput quantification of sphingoid bases from crude lipid extracts and is a valuable tool for studies of cellular sphingolipid metabolism and signaling.     

Quantitative profiling and pattern analysis of triacylglycerol species in Arabidopsis seeds by electrospray ionization mass spectrometry

Plant triacylglycerols (TAGs), or vegetable oils, provide approximately 25% of dietary calories to humans and are becoming an increasingly important source of renewable bioenergy and industrial feedstocks. TAGs are assembled by multiple enzymes in the endoplasmic reticulum from building blocks that include an invariable glycerol backbone and variable fatty acyl chains. It remains a challenge to elucidate the mechanism of synthesis of hundreds of different TAG species in planta. One reason is the lack of an efficient analytical approach quantifying individual molecular species. Here we report a rapid and quantitative TAG profiling approach for Arabidopsis seeds based on electrospray ionization tandem mass spectrometry with direct infusion and multiple neutral loss scans. The levels of 93 TAG molecular species, identified by their acyl components, were determined in Arabidopsis seeds. Quantitative TAG pattern analyses revealed that the TAG assembly machinery preferentially produces TAGs with one elongated fatty acid. The importance of the selectivity in oil synthesis was consistent with an observation that an Arabidopsis mutant overexpressing a patatin‐like phospholipase had enhanced seed oil content with elongated fatty acids. This quantitative TAG profiling approach should facilitate investigations aimed at understanding the biochemical mechanisms of TAG metabolism in plants.