Myeong Hee Moon

Field-flow fractionation in bioanalysis: A review of recent trends

Field-flow fractionation (FFF) is a mature technique in bioanalysis, and the number of applications to proteins and protein complexes, viruses, derivatized nano- and micronsized beads, sub-cellular units, and whole cell separation is constantly increasing. This can be ascribed to the non-invasivity of FFF when directly applied to biosamples. FFF is carried out in an open-channel structure by a flow stream of a mobile phase of any composition, and it is solely based on the interaction of the analytes with a perpendicularly applied field. For these reasons, fractionation is developed without surface interaction of the analyte with packing or gel media and without using degrading mobile phases. The fractionation device can be also easily sterilized, and analytes can be maintained under a bio-friendly environment. This allows to maintain native conditions of the sample in solution. In this review, FFF principles are briefly described, and some pioneering developments and applications in the bioanalytical field are tabled before detailed report of most recent FFF applications obtained also with the hyphenation of FFF with highly specific, sensitive characterization methods. Special focus is finally given to the emerging use of FFF as a pre-analytical step for mass-based identification and characterization of proteins and protein complexes in proteomics.

Proteomic Analysis of Exosomes from Human Neural Stem Cells by Flow Field-Flow Fractionation and Nanoflow Liquid Chromatography-Tandem Mass Spectrometry

Exosomes, small membrane vesicles secreted by a multitude of cell types, are involved in a wide range of physiological roles such as intercellular communication, membrane exchange between cells, and degradation as an alternative to lysosomes. Because of the small size of exosomes (30-100 nm) and the limitations of common separation procedures including ultracentrifugation and flow cytometry, size-based fractionation of exosomes has been challenging. In this study, we used flow field-flow fractionation (FlFFF) to fractionate exosomes according to differences in hydrodynamic diameter. The exosome fractions collected from FlFFF runs were examined by transmission electron microscopy (TEM) to morphologically confirm their identification as exosomes. Exosomal lysates of each fraction were digested and analyzed using nanoflow LC-ESI-MS-MS for protein identification. FIFFF, coupled with mass spectrometry, allows nanoscale size-based fractionation of exosomes and is more applicable to primary cells and stem cells since it requires much less starting material than conventional gel-based separation, in-gel digestion and the MS-MS method.

Shotgun lipidomics for candidate biomarkers of urinary phospholipids in prostate cancer

AbstractQualitative and quantitative profiling of six different categories of urinary phospholipids (PLs) from patients with prostate cancer was performed to develop an analytical method for the discovery of candidate biomarkers by shotgun lipidomics method. Using nanoflow liquid chromatography–electrospray ionization–tandem mass spectrometry, we identified the molecular structures of a total of 70 PL molecules (21 phosphatidylcholines (PCs), 11 phosphatidylethanolamines (PEs), 17 phosphatidylserines (PSs), 11 phosphatidylinositols (PIs), seven phosphatidic acids, and three phosphatidylglycerols) from urine samples of healthy controls and prostate cancer patients by data-dependent collision-induced dissociation. Identified molecules were quantitatively examined by comparing the MS peak areas. From statistical analyses, one PC, one PE, six PSs, and two PIs among the PL species showed significant differences between controls and cancer patients (p < 0.05, Student’s t test), with concentration changes of more than threefold. Cluster analysis of both control and patient groups showed that 18:0/18:1-PS and 16:0/22:6-PS were 99% similar in upregulation and that the two PSs (18:1/18:0, 18:0/20:5) with two PIs (18:0/18:1 and 16:1/20:2) showed similar (>95%) downregulation. The total amount of each PL group was compared among prostate cancer patients according to the Gleason scale as larger or smaller than 6. It proposes that the current study can be utilized to sort out possible diagnostic biomarkers of prostate cancer. FigureDendrogram of urinary phospholipids from prostate cancer

Heat-map visualization of gas chromatography-mass spectrometry based quantitative signatures on steroid metabolism

Abnormalities in steroid hormones are responsible for the development and prevention of endocrine diseases. Due to their biochemical roles in endocrine system, the quantitative evaluation of steroid hormones is needed to elucidate altered expression of steroids. Gas chromatographic-mass spectrometric (GC-MS) profiling of 70 urinary steroids, containing 22 androgens, 18 estrogens, 15 corticoids, 13 progestins, and 2 sterols, were validated and its quantitative data were visualized using hierarchically clustered heat maps to allow “steroid signatures”. The devised method provided a good linearity (r2 > 0.994) with the exception of cholesterol (r2 = 0.983). Precisions (% CV) and accuracies (% bias) ranged from 0.9% to 11.2% and from 92% to 119%, respectively, for most steroids tested. To evaluate metabolic changes, this method was applied to urine samples obtained from 59 patients with benign prostatic hyperplasia (BPH) versus 41 healthy male subjects. Altered concentrations of urinary steroids found and heat maps produced during this 70-compound study showed also differences between the ratios of steroid precursors and their metabolites (representing enzyme activity). Heat maps showed that oxidoreductases clustered (5β-reductase, 3β-HSD, 3β-HSD, and 17β-HSD, except for 20β-HSD). These results support that data transformation is valid, since 5β-reductase is a marker of BPH and 17β-HSD is positively expressed in prostate cells. Multitargeted profiling analysis of steroids generated quantitative results that help to explain correlations between enzyme activities. The data transformation and visualization described may to be found in the integration with the mining biomarkers of hormone-dependent diseases.

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.

Flow field-flow fractionation: A pre-analytical method for proteomics

Proteome analysis requires a comprehensive approach including high-performance separation methods, mass spectrometric analysis, and bioinformatics. While recent advances in mass spectrometry (MS) have led to remarkable improvements in the ability to characterize complex mixtures of biomolecules in proteomics, a proper pre-MS separation step of proteins/peptides is still required. The need of high-performance separation and/or isolation/purification techniques of proteins is increasing, due to the importance of proteins expressed at extremely low levels in proteome samples. In this review, flow field-flow fractionation (F4) is introduced as a complementary pre-analytical separation method for protein separation/isolation, which can be effectively utilized for proteomic research. F4 is a set of elution-based techniques that are capable of separating macromolecules by differences in diffusion coefficient and, therefore, in hydrodynamic size. F4 provides protein separation without surface interaction of the analyte with packing or gel media. Separation is carried out in an open channel structure by a flow stream of a mobile phase of any composition, and it is solely based on the interaction of the analytes with a perpendicularly-applied, secondary flow of the fluid. Therefore, biological analytes such as proteins can be kept under a bio-friendly environment without losing their original structural configuration. Moreover, proteins fractionated on a size/shape basis can be readily collected for further characterization or proteomic analysis by MS using, for instance, either on-line or off-line methods based on electrospray ionization (ESI) or matrix-assisted laser desorption-ionization (MALDI). This review focuses on the advantages of F4 compared to most-assessed separation/isolation techniques for proteomics, and on selected applications based on size-dependent proteome separation. New method developments based on the hyphenation of F4 with on-line or off-line MS, and with other separation methods such as capillary isoelectric focusing (CIEF) are also described.

Effect of viscosity on retention time and hydrodynamic lift forces in sedimentation/steric field-flow fractionation

Abstract Particles entrained in fluid flow between parallel bounding walls tend to be driven by hydrodynamic forces, acting perpendicular to the direction of flow, towards certain equilibrium positions between the walls. Sedimentation field-flow fractionation is a technique that is well suited to the measurement of these forces, particularly in the regions close to the walls. Forces much stronger than those due to fluid inertial effects are commonly observed in the near-wall regions. A study is presented here of the influence of carrier fluid viscosity on these hydrodynamic lift forces. The carrier viscosity is varied via two different methods: (1) various ternary mixtures of water, glycerol, and ethanol were used that varied in viscosity while being of constant density and (2) the temperature of the FFF system was raised, changing the carrier viscosity while not significantly altering its density. The near-wall lift forces are shown to be dependent on fluid viscosity. An empirical equation describing the apparent dependence is presented.

Hyperlayer hollow-fiber flow field-flow fractionation of cells.

Interest in low-cost, analytical-scale, highly efficient and sensitive separation methods for cells, among which bacteria, is increasing. Particle separation in hollow-fiber flow field-flow fractionation (HF FlFFF) has been recently improved by the optimization of the HF FIFFF channel design. The intrinsic simplicity and low cost of this HF FlFFF channel allows for its disposable usage. which is particularly appealing for analytical bio-applications. Here, for the first time, we present a feasibility study on high-performance, hyperlayer HF FIFFF of micrometer-sized bacteria (Escherichia coli) and of different types of cells (human red blood cells, wine-making yeast from Saccharomyces cerevisiae). Fractionation performance is shown to be at least comparable to that obtained with conventional, flat-channel hyperlayer FIFFF of cells, at superior size-based selectivity and reduced analysis time.

Size characterization of liposomes by flow field-flow fractionation and photon correlation spectroscopy: Effect of ionic strength and pH of carrier solutions

The effect of ionic strength and pH of carrier solutions on the separation of liposomes by flow field-flow fractionation (flow FFF) has been studied for the determination of accurate vesicle size distribution of liposomes. Retention behaviors of liposomes (PC/PG/cholesterol) are observed in typical buffer solutions (PBS and Tris-HCl) of various ionic strengths as carrier liquids in flow FFF. The average diameters of collected fractions at each flow FFF run are measured by photon correlation spectroscopy (PCS) for the comparison with FFF calculations at corresponding time interval of collected fractions. A reasonable separation of liposomes is observed at I = 0.016 M for both buffer solutions. Retention of liposomes is found to be elongated at ionic strengths higher than an optimum condition found experimentally, but it is shortened at a lower ionic strength due to the electrostatic interaction between the channel wall and the liposomes. Finally, size distributions of liposomes are provided comparing the liposome preparations by flow FFF.

Size distribution of liposomes by flow field-flow fractionation.

The applicability of field-low fractionation (FFF) to the characterization of liposomes is discussed and theoretically described. Because of fundamental differences in their driving forces, sedimentation FFF and flow FFF measure different vesicle properties. Sedimentation FFF, although used previously to measure vesicle sizes and size distributions, is fundamentally a technique that measures the effective mass and mass distribution of particles. It is sensitive to small changes in the effective mass of either the biomembrane or its encapsulated load and thus is likely to be useful in characterizing such properties as drug loading, biomembrane volumes and areas, and distributions of these properties. Size characterization by sedimentation FFF can only be done by deducing size from effective mass. Flow FFF, by contrast, provides a direct measurement of vesicle size and size distribution. After demonstrating the high resolution and relative accuracy of size measurement of flow FFF by the separation of polystyrene latex standards, flow FFF was applied to two preparations of DSPC-DSPA liposomes that were sonicated under different temperature conditions. Fractograms and size distributions are reported as a function of sonication time. The rapid elimination of a large diameter tail to the distribution is shown to constitute a major mechanism for distribution narrowing. Finally, results are provided bearing on the reproducibility of size distribution measurements by flow FFF.