Ju Yong Lee

Simultaneous profiling of lysophospholipids and phospholipids from human plasma by nanoflow liquid chromatography-tandem mass spectrometry

AbstractIn this study, an analytical method for the simultaneous separation and characterization of various molecular species of lysophospholipids (LPLs) and phospholipids (PLs) is introduced by employing nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). Since LPLs and PLs in human plasma are potential biomarkers for cancer, development of a sophisticated analytical method for the simultaneous profiling of these molecules is important. Standard species of LPLs and PLs were examined to establish a separation condition using a capillary LC column followed by MS scans and data-dependent collision-induced dissociation (CID) analysis for structural identification. With nLC-ESI-MS/MS, regioisomers of each category of LPLs were completely separated and identified with characteristic CID spectra. It was applied to the comprehensive profiling of LPLs and PLs from a human blood plasma sample and yielded identifications of 50 LPLs (each regioisomer pair of 6 lysophosphatidylcholines (LPCs), 7 lysophosphatidylethanolamines (LPEs), 9 lysophosphatidic acid (LPAs), 2 lysophosphatidylglycerols (LPGs), and 1 lysophosphatidylserine (LPS)) and 62 PLs (19 phosphatidylcholines (PCs), 11 phosphatidylethanolamines (PEs), 3 phosphatidylserines (PSs), 16 phosphatidylinositols (PIs), 8 phosphatidylglycerols (PGs), and 5 phosphatidic acids (PAs)). FigureThe study demonstrates that regioisomers of lysophospholipid can be completely separated and identified with characteristic CID spectra using nLC-ESI-MS-MS, along with the simultaneous profiling of phospholipids from human blood plasma.

Profiling of phospholipids in lipoproteins by multiplexed hollow fiber flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry.

This study describes a coupled analytical method to carry out the systematic profiling of phospholipids (PLs) in high-density lipoproteins (HDL) and low-density lipoproteins (LDL) from human blood plasma. HDL and LDL of healthy human plasma samples were separated by size and collected on a semi-preparative scale using multiplexed hollow fiber flow field-flow fractionation (MxHF5). Phospholipid mixtures contained in the resulting HDL and LDL fractions were analyzed by shotgun nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS-MS). We utilized a dual scan method for the separation and simultaneous characterization of complicated PL mixtures by nLC-ESI-MS-MS, such that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules were detected in positive ion mode in a first LC run. In a second LC run, phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA) were detected in negative ion mode. In this study, a total of 56 PLs from HDL and 52 PLs from LDL particles were characterized by their molecular structures from data dependent collision-induced dissociation (CID) experiments, and their relative abundances were compared.

Lipidomic profiling of plasma and urine from patients with Gaucher disease during enzyme replacement therapy by nanoflow liquid chromatography–tandem mass spectrometry

Gaucher disease (GD) is a rare genetic disorder that arises from lipid species, especially monohexosylceramide (MHC), accumulating in different organs. GD results from a β-glucocerebrosidase deficiency, causing metabolic or neurologic complications. This study comprehensively profiled lipids from patients and healthy controls to discover active lipid species related to GD. Most studies have evaluated lipids from one type of biological sample, such as plasma, urine, or spinal fluid, which are the main sources of lipids in human bodies. The purpose of this study, however, was to collect and assess both plasma and urine samples from a group of individuals, explore the lipids, and select characteristic species that show significant differences between controls and patients from the two sources. Also, the response of lipids to enzyme replacement therapy (ERT), which is targeted to reduce excessive lipid accumulation within lysosomes, was investigated by obtaining plasma and urine from patients after receiving the therapy. Most lipid species were found in both plasma and urine but their concentrations differed, and some species were found in either plasma or urine only. Out of 125 plasma and 105 urinary lipids that were identified by nLC-ESI-MS/MS, 20 plasma and 10 urinary lipids were selected as characteristic species for having average concentrations that were significantly increased or decreased in patients by greater than 2-fold. Moreover, the concentrations of most lipids that showed greater than 2-fold of difference in patients decreased after ERT indicating that these species were directly or indirectly affected by the therapy.

Computational approach to structural identification of phospholipids using raw mass spectra from nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry.

A qualitative analysis tool (LiPilot) for identifying phospholipids (PLs), including lysophospholipids (LPLs), from biological mixtures is introduced. The developed algorithm utilizes raw data obtained from nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry experiments of lipid mixture samples including retention time and m/z values of precursor and fragment ions from data-dependent, collision-induced dissociation. Library files based on typical fragmentation patterns of PLs generated with an LTQ-Velos ion trap mass spectrometer are used to identify PL or LPL species by comparing experimental fragment ions with typical fragment ions in the library file. Identification is aided by calculating a confidence score developed in our laboratory to maximize identification efficiency. Analysis includes the influence of total ion intensities of matched and unmatched fragment ions, the difference in m/z values between observed and theoretical fragment ions, and a weighting factor used to differentiate regioisomers through data filtration. The present study focused on targeted identification of particular PL classes. The identification software was evaluated using a mixture of 24 PL and LPL standards. The software was further tested with a human urinary PL mixture sample, with 93 PLs and 22 LPLs identified.

Profiling of Oxidized Phospholipids in Lipoproteins from Patients with Coronary Artery Disease by Hollow Fiber Flow Field-Flow Fractionation and Nanoflow Liquid Chromatography–Tandem Mass Spectrometry

Oxidized phospholipids (Ox-PLs) are oxidatively modified PLs that are produced during the oxidation of lipoproteins; oxidation of low density lipoproteins especially is known to be associated with the development of coronary artery disease (CAD). In this study, different lipoprotein classes (high density, low density, and very low density lipoproteins) from pooled plasma of CAD patients and pooled plasma from healthy controls were size-sorted on a semipreparative scale by multiplexed hollow fiber flow field-flow fractionation (MxHF5), and Ox-PLs that were extracted from each lipoprotein fraction were quantified by nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS). The present study showed that oxidation of lipoproteins occurred throughout all classes of lipoproteins with more Ox-PLs identified from CAD patient lipoproteins: molecular structures of 283 unique PL species (including 123 Ox-PLs) from controls and 315 (including 169 Ox-PLs) from patients were identified by data-dependent collision-induced dissociation experiments. It was shown that oxidation of PLs occurred primarily with hydroxylation of PL; in particular, a saturated acyl chain such as 16:0, 18:0, or even 18:1 at the sn-1 location of the glycerol backbone along with sn-2 acyl chains with at least two double bonds were identified. The acyl chain combinations commonly found for hydroxylated Ox-PLs in the lipoproteins of CAD patients were 16:0/18:2, 16:0/20:4, 18:0/18:2, and 18:0/20:4.

Discovery of candidate phospholipid biomarkers in human lipoproteins with coronary artery disease by flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry.

In this study, an analytical method is demonstrated to identify and develop potential phospholipid (PL) biomarkers of high density lipoprotein (HDL) and low density lipoprotein (LDL) in plasma from individuals with coronary artery disease (CAD) by employing a combination of off-line multiplexed hollow fiber flow field-flow fractionation (MxHF5) and nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). HDL and LDL particles of human plasma were sorted by size at a semi-preparative scale using MxHF5, after which PL extracts of each lipoprotein fraction were qualitatively and quantitatively analyzed by nLC-ESI-MS-MS. Experiments were performed using plasma samples from 10 CAD patients and 10 controls. Quantitative analysis of the 93 PL species identified yielded a selection of 19 species from HDL fractions and 10 from LDL fractions exhibiting at least a five fold change in average concentration in CAD patients. Among the selected species, only a few were found exclusively in patient HDL fractions (18:3-LPA and 20:2/16:0-PG), control HDL fractions (16:0/16:1-PC, 20:1/20:4-PE, and 16:1-LPA), and control LDL fractions (16:0/22:3-PG). Moreover, 16:1/18:2-PC was detected from both HDL and LDL fractions of controls but disappeared in CAD patients. Although the typical change in lipoproteins for CAD is well known, with decreased levels of HDLs and reduced LDL particle size, the current study provides fundamental information on the molecular level of lipoprotein variation which can be utilized for diagnostic and therapeutic tracking.

Effect of asymmetrical flow field-flow fractionation channel geometry on separation efficiency

The separation efficiencies of three different asymmetrical flow field-flow fractionation (AF4) channel designs were evaluated using polystyrene latex standards. Channel breadth was held constant for one channel (rectangular profile), and was reduced either linearly (trapezoidal profile) or exponentially (exponential profile) along the length for the other two. The effective void volumes of the three channel types were designed to be equivalent. Theoretically, under certain flow conditions, the mean channel flow velocity of the exponential channel could be arranged to remain constant along the channel length, thereby improving separation in AF4. Particle separation obtained with the exponential channel was compared with particle separation obtained with the trapezoidal and rectangular channels. We demonstrated that at a certain flow rate condition (outflow/inflow rate=0.2), the exponential channel design indeed provided better performance with respect to the separation of polystyrene nanoparticles in terms of reducing band broadening. While the trapezoidal channel exhibited a little poorer performance than the exponential, the strongly decreasing mean flow velocity in the rectangular channel resulted in serious band broadening, a delay in retention time, and even failure of larger particles to elute.

Top-down lipidomic analysis of human lipoproteins by chip-type asymmetrical flow field-flow fractionation-electrospray ionization-tandem mass spectrometry

This study demonstrates the potential utility of on-line chip-type asymmetrical flow field-flow fractionation (cAF4) and electrospray ionization tandem mass spectrometry (ESI-MS-MS) for the top-down lipidomic analysis of human lipoproteins. Utilizing a cAF4, which is a miniaturized AF4 channel operated with a micro flow rate regime, enabled high density lipoprotein (HDL) and low density lipoprotein (LDL) to be separated by hydrodynamic diameter in an aqueous solution with the simultaneous desalting of lipoproteins. On-line desalting was found to enhance the ionization of lipoproteinic lipid molecules during the feeding of cAF4 effluent to ESI-MS when compared to the direct infusion of lipoproteins to MS. An evaluation of top-down lipidomic analysis was performed to test the efficiency of in-source fragmentation during cAF4-ESI-MS in the dissociation of lipoprotein particles into individual lipid molecules. This study demonstrates the structural identification of the following lipid classes: phosphatidylcholines (PCs), cholesteryl esters (CEs), and regioisomers of triacylglycerols (TAGs) having an identical mass but different acyl chains and dimeric forms of TAGs in the positive ion mode, and phosphatidylglycerols (PGs), phosphatidic acids (PAs), phosphatidyinositols (PIs), and their lyso species in the negative ion mode. The developed method was applied to plasma samples from patients with coronary artery disease (CAD) for the separation of HDL and LDL and for the simultaneous analysis of lipoproteinic lipids, resulting in the identification of 11 PCs, 9 PGs, 4 PAs, 2 PIs, 2 PEs, 18 TAGs, and 6 CEs.

Evaluation of multiplexed hollow fiber flow field-flow fractionation for semi-preparative purposes

A multiplexed hollow fiber flow field-flow fractionation (MxHF5) is introduced to increase throughput of an HF5 channel system for semi-preparative purposes. HF5, a cylindrical version of the flow field-flow fractionation (FlFFF) operated with a porous, hollow fiber membrane by controlling the ratio of radial and axial flow rates, is capable of fractionating proteins, cells, and macromolecules by size. An advantage of HF5 is its inexpensive channel construction, allowing for disposability that can reduce run-to-run carryover problems. MxHF5 constructed in this study was made with six parallel HF5 modules connected to seven-port manifolds for the semi-preparative scale separation of proteins or biological particles. For the evaluation of MxHF5 separation efficiency, protein standards were utilized to test peak recoveries, band broadening, and throughput. The assembly showed the possibility of handling up to 50 microg of proteins without incurring overloading. The developed channel was applied to demonstrate size sorting of lipoproteins for the future study of size dependent lipidomic and proteomic analysis. The current trial offers a unique advantage of scaling up HF5 separation without using wide-bore, hollow fibers which sacrifice separation speed.

On-line miniaturized asymmetrical flow field-flow fractionation-electrospray ionization-tandem mass spectrometry with selected reaction monitoring for quantitative analysis of phospholipids in plasma lipoproteins

A direct analytical method for high speed quantitative analysis of lipids in human blood plasma using on-line chip-type asymmetrical flow field-flow fractionation-electrospray ionization-tandem mass spectrometry (cAF4-ESI-MS/MS) with selected reaction monitoring (SRM) is described in this study. Utilizing a miniaturized cAF4 channel, high speed size separation of high density lipoproteins (HDL) and low density lipoproteins (LDL) from plasma samples can be accomplished at a microflow rate along with simultaneous desalting of lipoproteins, both of which are conducive to direct ESI of lipids in lipoproteins. This study demonstrates that the SRM method to monitor phospholipids during cAF4-ESI-MS/MS can be successfully applied to the quantitation of lipid molecules in plasma lipoproteins without the need of a separate lipid extraction process. For quantitation of lipids in HDL and LDL during cAF4-ESI-MS/MS runs, a protein standard (carbonic anhydrase, 29 kDa) was added to each plasma sample as an internal standard such that a peak intensity of y67(+5) ions, which are high abundant SRM product ions of CA, could be utilized to calculate the relative intensity of each lipid molecule. The developed method was applied to plasma samples from 10 patients with coronary artery disease (CAD) and 10 healthy control samples, and quantitative analysis of 39 lipid molecules including phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, phosphatidylglycerols, and phosphatidylinositols, resulted in the selection of 13 PL species showing more than 2.5 fold difference in relative abundance (p<0.01) between the groups. The present study demonstrates a high speed analytical method for determining plasma lipid content and distribution without an organic solvent extraction of lipids from plasma.