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Article ; Online: Comparison of a thickness-tapered channel in flow field-flow fractionation with a conventional channel with flow rate programming.

Kim, Young Beom / Kim, Jaihoo / Williams, P Stephen / Moon, Myeong Hee

2024 Volume 1724, Page(s) 464927

Abstract: The thickness-tapered channel structure in flow field-flow fractionation (FlFFF), recently introduced by constructing a channel with a linear decrease in thickness along its length, demonstrated effectiveness in steric/hyperlayer separation of ... ...

Abstract	The thickness-tapered channel structure in flow field-flow fractionation (FlFFF), recently introduced by constructing a channel with a linear decrease in thickness along its length, demonstrated effectiveness in steric/hyperlayer separation of supramicron particles with improvements in separation speed, elution recovery, and an expanded dynamic size range of separation. In this study, we conducted a comparative analysis of the performance between the impact of field (or crossflow rate) programming or outflow rate programming for the separation of polystyrene latex standards (50 ∼ 800 nm) with a conventional channel having uniform thickness and a thickness-tapered channel without programming. Outlet flow rate and crossflow rate conditions were also varied. Although the particle size resolution of the tapered channel does not surpass that of field programming in uniform thickness channel, it achieves higher-speed separation without a significant loss of resolution and without the need for a complex flow controller system even at a low outflow rate condition. Furthermore, it yielded an improved resolution for particles close to the steric transition regime (400 ∼ 600 nm) in the normal mode of separation. Due to the continuous increase in mean flow velocity down the channel, the tapered channel exhibits flexibility in separating submicron-sized particles at high crossflow rate conditions or low outflow rate conditions, of which the latter can be advantageous when coupled with mass spectrometry in a miniaturized setup.
MeSH term(s)	Fractionation, Field Flow/methods ; Particle Size ; Polystyrenes/chemistry ; Equipment Design
Chemical Substances	Polystyrenes
Language	English
Publishing date	2024-04-20
Publishing country	Netherlands
Document type	Journal Article ; Comparative Study
ZDB-ID	1171488-8
ISSN	1873-3778 ; 0021-9673
ISSN (online)	1873-3778
ISSN	0021-9673
DOI	10.1016/j.chroma.2024.464927
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Article: Flow field-flow fractionation: Recent applications for lipidomic and proteomic analysis

Moon, Myeong Hee

Trends in analytical chemistry. 2019 Sept., v. 118

2019

Abstract: Flow field-flow fractionation (FlFFF) is a versatile size-based separation method suitable for biological macromolecules including proteins/protein aggregates, DNA, subcellular organelles, extracellular species, and whole cells. This review introduces ... ...

Abstract	Flow field-flow fractionation (FlFFF) is a versatile size-based separation method suitable for biological macromolecules including proteins/protein aggregates, DNA, subcellular organelles, extracellular species, and whole cells. This review introduces briefly the basic principles of FlFFF and its recent applications for proteomic and lipidomic analysis, which are described in two parts: (1) off-line coupling of FlFFF with MS and other bioanalytical methods, and (2) on-line FlFFF with MS. The first part includes applications for lipoproteins, exosomes, and subcellular organelles for the size-dependent analysis of proteins and lipids in narrow size-fractions collected during FlFFF, followed by independent analysis including western blotting and nanoflow liquid chromatography-electrospray ionisation-tandem mass spectrometry (nLC-ESI-MS/MS). The second part highlights the on-line FlFFF-MS, in which a miniaturised FlFFF channel is coupled to ESI-MS/MS for the high-speed lipid analysis of lipoproteins and to inductively-coupled plasma MS for the direct analysis of metals in metalloproteins from blood plasma.
Keywords	DNA ; Western blotting ; blood plasma ; exosomes ; fractionation ; lipoproteins ; liquid chromatography ; metalloproteins ; metals ; protein aggregates ; proteomics ; tandem mass spectrometry
Language	English
Dates of publication	2019-09
Size	p. 19-28.
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	2014041-1
ISSN	0165-9936
ISSN	0165-9936
DOI	10.1016/j.trac.2019.05.024
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Article ; Online: Size Separation of Exosomes and Microvesicles Using Flow Field-Flow Fractionation/Multiangle Light Scattering and Lipidomic Comparison.

Kim, Young Beom / Lee, Gwang Bin / Moon, Myeong Hee

Analytical chemistry

2022 Volume 94, Issue 25, Page(s) 8958–8965

Abstract: Extracellular vesicles (EVs) are cell-derived membrane-bound particles, including exosomes and microvesicles that differ in cellular origin, content, and lipid composition. This study reports that exosomes and microvesicles can be simultaneously ... ...

Abstract	Extracellular vesicles (EVs) are cell-derived membrane-bound particles, including exosomes and microvesicles that differ in cellular origin, content, and lipid composition. This study reports that exosomes and microvesicles can be simultaneously separated by size using flow field-flow fractionation (FlFFF) employed with field programming and that the detection of low-concentration EV species can be significantly improved using multiangle light scattering (MALS). The efficiency of ultracentrifugation (UC) and ultrafiltration (UF) in isolating EVs from the culture media of DU145 cells was compared, and the results showed that UF retrieves more EVs than UC. Two size fractions (small and large) of both exosomes and microvesicles were collected during the FlFFF runs and examined using Western blotting to confirm each EV, and transmission electron microscopy was performed for size analysis. Sizes were compared using the root-mean-square radius obtained from the MALS calculation. The collected fractions were further examined using nanoflow ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the size-dependent lipidomic profiles of exosomes and microvesicles, showing that lipids were more enriched in the fraction containing large exosomes than in that containing small exosomes; however, an opposite trend was observed with microvesicles. The present study demonstrated that UF followed by FlFFF-MALS can be utilized for the size separation of exosomes and microvesicles without sequential centrifugation, which is useful for monitoring the changes in the size distribution of EVs depending on the biological status along with generating size-dependent lipidomic profiles.
MeSH term(s)	Cell-Derived Microparticles ; Exosomes/chemistry ; Fractionation, Field Flow/methods ; Lipidomics ; Ultracentrifugation
Language	English
Publishing date	2022-06-13
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.2c00806
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Article ; Online: Correction to Relative Quantification of Phospholipids Based on Isotope-Labeled Methylation by Nanoflow Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry: Enhancement in Cardiolipin Profiling.

Lee, Jong Cheol / Byeon, Seul Kee / Moon, Myeong Hee

Analytical chemistry

2021 Volume 93, Issue 19, Page(s) 7378–7379

Language	English
Publishing date	2021-05-07
Publishing country	United States
Document type	Published Erratum
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.1c01819
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Article ; Online: Corrigendum to "Flow optimisations with increased channel thickness in asymmetrical flow field-flow fractionation" [J. Chromatogr. A 1581-1582 (2018) 100-104].

Yang, Joon Seon / Moon, Myeong Hee

Journal of chromatography. A

2019 Volume 1591, Page(s) 178

Language	English
Publishing date	2019-03-13
Publishing country	Netherlands
Document type	Journal Article ; Published Erratum
ZDB-ID	1171488-8
ISSN	1873-3778 ; 0021-9673
ISSN (online)	1873-3778
ISSN	0021-9673
DOI	10.1016/j.chroma.2019.03.013
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Article ; Online: Optimisation of saliva volumes for lipidomic analysis by nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry.

Lee, Gwang Bin / Caner, Ayse / Moon, Myeong Hee

Analytica chimica acta

2021 Volume 1193, Page(s) 339318

Abstract: Saliva is a readily accessible and clinically useful biofluid that can be used to develop disease biomarkers because of a variety of biologically active molecules in it that are also found in blood. However, even though saliva sampling is simple and non- ... ...

Abstract	Saliva is a readily accessible and clinically useful biofluid that can be used to develop disease biomarkers because of a variety of biologically active molecules in it that are also found in blood. However, even though saliva sampling is simple and non-invasive, few studies have investigated the use of salivary lipids as biomarkers, and the extraction of lipids from saliva needs to be examined thoroughly. In the present study, methods (i.e., saliva sample volume, 0.1-1.0 mL) for the extraction and analysis of salivary lipids by nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry (nUHPLC-ESI-MS/MS) were evaluated according to the matrix effect, extraction recovery, and number of quantifiable lipids. A total of 780 lipids were identified in a pooled saliva sample from 20 healthy volunteers, and 372 lipids without differentiating acyl chain structures were quantified, along with comprehensive information on salivary lipid composition and individual lipid levels. Even though extraction recovery was maintained at saliva sample volumes as low as 0.2 mL, the matrix effect and limit of detection (LOD) were relatively large with 1.0 mL. Considering the matrix effect, LOD, and number of quantifiable lipids (>limit of quantitation), the minimum volume of saliva sufficient for lipidomic analysis using nUHPLC-ESI-MS/MS was determined to be 0.5 mL.
MeSH term(s)	Chromatography, High Pressure Liquid ; Humans ; Lipidomics ; Lipids ; Saliva ; Tandem Mass Spectrometry
Chemical Substances	Lipids
Language	English
Publishing date	2021-11-23
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	1483436-4
ISSN	1873-4324 ; 0003-2670
ISSN (online)	1873-4324
ISSN	0003-2670
DOI	10.1016/j.aca.2021.339318
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Article ; Online: Enhancement of acidic lipid analysis by nanoflow ultrahigh performance liquid chromatography-mass spectrometry.

Lee, Jong Cheol / Kim, Young Beom / Moon, Myeong Hee

Analytica chimica acta

2021 Volume 1166, Page(s) 338573

Abstract: Acidic lipids are associated with the regulation of the structure and function of membrane proteins. Therefore, accurate and highly precise analysis of acidic lipids is important for elucidating their biological roles and pathological mechanisms. In this ...

Abstract	Acidic lipids are associated with the regulation of the structure and function of membrane proteins. Therefore, accurate and highly precise analysis of acidic lipids is important for elucidating their biological roles and pathological mechanisms. In this study, an enhanced analytical method for the separation and quantification of acidic lipids, including phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin, and their lyso-derivatives, was developed using nanoflow ultrahigh performance liquid chromatography-electrospray ionisation-tandem mass spectrometry. The separation and mass spectrometry detection of acidic lipids were optimised in terms of peak tailing and time-based separation efficiencies, with carbamate-embedded C18 as the stationary phase, in the presence of an appropriate liquid chromatography solvent modifier. This newly developed method was applied to analyse a lipid extract from porcine brain. A significant increase in the number of acidic lipids identified (176 vs. 134), including intact monolysocardiolipin (17 vs. 4), was observed with the new method compared with conventional C18. The quantification of acidic lipids was validated with plasma standard (NIST SRM 1950) spiked with a number of LPS and PS standards, and acceptable accuracy (<15%) was obtained. The present method was found to be reliable for the acidic lipid analysis based on qualitative results from tissue extract and plasma samples.
MeSH term(s)	Animals ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Mass Spectrometry ; Plasma ; Solvents ; Swine
Chemical Substances	Solvents
Language	English
Publishing date	2021-04-27
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	1483436-4
ISSN	1873-4324 ; 0003-2670
ISSN (online)	1873-4324
ISSN	0003-2670
DOI	10.1016/j.aca.2021.338573
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Article: Size Separation of Exosomes and Microvesicles Using Flow Field-Flow Fractionation/Multiangle Light Scattering and Lipidomic Comparison

Kim, Young Beom / Lee, Gwang Bin / Moon, Myeong Hee

Analytical chemistry. 2022 June 13, v. 94, no. 25

2022

Abstract	Extracellular vesicles (EVs) are cell-derived membrane-bound particles, including exosomes and microvesicles that differ in cellular origin, content, and lipid composition. This study reports that exosomes and microvesicles can be simultaneously separated by size using flow field-flow fractionation (FlFFF) employed with field programming and that the detection of low-concentration EV species can be significantly improved using multiangle light scattering (MALS). The efficiency of ultracentrifugation (UC) and ultrafiltration (UF) in isolating EVs from the culture media of DU145 cells was compared, and the results showed that UF retrieves more EVs than UC. Two size fractions (small and large) of both exosomes and microvesicles were collected during the FlFFF runs and examined using Western blotting to confirm each EV, and transmission electron microscopy was performed for size analysis. Sizes were compared using the root-mean-square radius obtained from the MALS calculation. The collected fractions were further examined using nanoflow ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the size-dependent lipidomic profiles of exosomes and microvesicles, showing that lipids were more enriched in the fraction containing large exosomes than in that containing small exosomes; however, an opposite trend was observed with microvesicles. The present study demonstrated that UF followed by FlFFF-MALS can be utilized for the size separation of exosomes and microvesicles without sequential centrifugation, which is useful for monitoring the changes in the size distribution of EVs depending on the biological status along with generating size-dependent lipidomic profiles.
Keywords	analytical chemistry ; exosomes ; fractionation ; lipid composition ; lipidomics ; transmission electron microscopy ; ultracentrifugation ; ultrafiltration
Language	English
Dates of publication	2022-0613
Size	p. 8958-8965.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.2c00806
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Article ; Online: Flow optimisations with increased channel thickness in asymmetrical flow field-flow fractionation.

Yang, Joon Seon / Moon, Myeong Hee

Journal of chromatography. A

2018 Volume 1581-1582, Page(s) 100–104

Abstract: Retention in flow field-flow fractionation (flow FFF) is generally governed by the combination of crossflow and migration flowrates. Especially for an asymmetrical flow FFF (AF4) channel in which the channel-inlet flow is divided into crossflow and ... ...

Abstract	Retention in flow field-flow fractionation (flow FFF) is generally governed by the combination of crossflow and migration flowrates. Especially for an asymmetrical flow FFF (AF4) channel in which the channel-inlet flow is divided into crossflow and outflow, the separation of low-molecular-weight proteins or macromolecules requires a relatively high crossflow rate along with a very low outflow rate for a reasonable level of resolution, which often leads to a limitation in channel pressure. In this study, the performances of AF4 with increased channel thicknesses have been investigated by adjusting the effective channel flowrates in the asymmetrical channels according to the variation of channel thickness. Four AF4 channels of different channel thicknesses (350, 490, 600, and 740 μm) were employed to examine the potential usefulness of employing a thick channel in the high-resolution separation of low-molecular-weight proteins (< 100 kDa) and to determine the relationship between higher channel thickness and the recovery of elution. Experiments showed that the ratio of crossflow rate to the effective channel flowrate should be considered in the selection of a run condition at an increased channel thickness. The study also demonstrated that a thick AF4 channel can be useful for the high-resolution separation of low-molecular-weight species such as protein aggregates without using extremely high crossflow rates.
MeSH term(s)	Chemistry Techniques, Analytical/methods ; Fractionation, Field Flow ; Molecular Weight
Language	English
Publishing date	2018-10-31
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	1171488-8
ISSN	1873-3778 ; 0021-9673
ISSN (online)	1873-3778
ISSN	0021-9673
DOI	10.1016/j.chroma.2018.10.053
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Article ; Online: High-Speed Screening of Lipoprotein Components Using Online Miniaturized Asymmetrical Flow Field-Flow Fractionation and Electrospray Ionization Tandem Mass Spectrometry: Application to Hepatocellular Carcinoma Plasma Samples.

Kim, Jin Yong / Lee, Gwang Bin / Lee, Jong Cheol / Moon, Myeong Hee

Analytical chemistry

2021 Volume 93, Issue 11, Page(s) 4867–4875

Abstract: This study introduces a high-speed screening method for the quantitative analysis of lipoprotein components in human plasma samples using online miniaturized asymmetrical flow field-flow fractionation and electrospray ionization-tandem mass spectrometry ( ...

Abstract	This study introduces a high-speed screening method for the quantitative analysis of lipoprotein components in human plasma samples using online miniaturized asymmetrical flow field-flow fractionation and electrospray ionization-tandem mass spectrometry (mAF4-ESI-MS/MS). Using an mAF4 channel, high-density lipoproteins and low-density lipoproteins can be fractionated by size at a high speed (<10 min) and directly fed to ESI-MS/MS for the simultaneous screening of targeted lipid species and apolipoprotein A1 (ApoA1). By employing the heated electrospray ionization probe as an ionization source, an mAF4 effluent flow rate of up to a few tens of microliters per minute can be used, which is adequate for direct feeding to MS without splitting the outflow, resulting in a consistent feed rate to MS for stable MS detection. mAF4-ESI-MS/MS was applied to hepatocellular carcinoma (HCC) plasma samples for targeted quantification of 25 lipid biomarker candidates and ApoA1 compared with healthy controls, the results of which were in statistical agreement with the quantified results obtained by nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry. Moreover, the present method provided the simultaneous detection of changes in lipoprotein size and the relative amount. This study demonstrated the potential of mAF4-ESI-MS/MS as an alternative high-speed screening platform for the top-down analysis of targeted lipoprotein components in patients with HCC, which is applicable to other diseases that involve the perturbation of lipoproteins.
MeSH term(s)	Carcinoma, Hepatocellular ; Humans ; Lipoproteins ; Liver Neoplasms/diagnosis ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry
Chemical Substances	Lipoproteins
Language	English
Publishing date	2021-03-10
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1508-8
ISSN	1520-6882 ; 0003-2700
ISSN (online)	1520-6882
ISSN	0003-2700
DOI	10.1021/acs.analchem.0c04756
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