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

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.

Comprehensive Lipid Profiling Recapitulates Enhanced Lipolysis and Fatty Acid Metabolism in Intimal Foamy Macrophages From Murine Atherosclerotic Aorta.

Lipid accumulation in macrophages is a prominent phenomenon observed in atherosclerosis. Previously, intimal foamy macrophages (FM) showed decreased inflammatory gene expression compared to intimal non-foamy macrophages (NFM). Since reprogramming of lipid metabolism in macrophages affects immunological functions, lipid profiling of intimal macrophages appears to be important for understanding the phenotypic changes of macrophages in atherosclerotic lesions. While lipidomic analysis has been performed in atherosclerotic aortic tissues and cultured macrophages, direct lipid profiling has not been performed in primary aortic macrophages from atherosclerotic aortas. We utilized nanoflow ultrahigh-performance liquid chromatography-tandem mass spectrometry to provide comprehensive lipid profiles of intimal non-foamy and foamy macrophages and adventitial macrophages from Ldlr-/- mouse aortas. We also analyzed the gene expression of each macrophage type related to lipid metabolism. FM showed increased levels of fatty acids, cholesterol esters, phosphatidylcholine, lysophosphatidylcholine, phosphatidylinositol, and sphingomyelin. However, phosphatidylethanolamine, phosphatidic acid, and ceramide levels were decreased in FM compared to those in NFM. Interestingly, FM showed decreased triacylglycerol (TG) levels. Expressions of lipolysis-related genes including Pnpla2 and Lpl were markedly increased but expressions of Lpin2 and Dgat1 related to TG synthesis were decreased in FM. Analysis of transcriptome and lipidome data revealed differences in the regulation of each lipid metabolic pathway in aortic macrophages. These comprehensive lipidomic data could clarify the phenotypes of macrophages in the atherosclerotic aorta.

Salivary Lipids of Patients with Non-Small Cell Lung Cancer Show Perturbation with Respect to Plasma.

A comprehensive lipid profile was analyzed in patients with non-small cell lung cancer (NSCLC) using nanoflow ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. This study investigated 297 and 202 lipids in saliva and plasma samples, respectively, comparing NSCLC patients to healthy controls. Lipids with significant changes (>2-fold, p < 0.05) were further analyzed in each sample type. Both saliva and plasma exhibited similar lipid alteration patterns in NSCLC, but saliva showed more pronounced changes. Total triglycerides (TGs) increased (>2-3-fold) in plasma and saliva samples. Three specific TGs (50:2, 52:5, and 54:6) were significantly increased in NSCLC for both sample types. A common ceramide species (d18:1/24:0) and phosphatidylinositol 38:4 decreased in both plasma and saliva by approximately two-fold. Phosphatidylserine 36:1 was selectively detected in saliva and showed a subsequent decrease, making it a potential biomarker for predicting lung cancer. We identified 27 salivary and 10 plasma lipids as candidate markers for NSCLC through statistical evaluations. Moreover, this study highlights the potential of saliva in understanding changes in lipid metabolism associated with NSCLC.

Impaired BCAA catabolism in adipose tissues promotes age-associated metabolic derangement.

Adipose tissues are central in controlling metabolic homeostasis and failure in their preservation is associated with age-related metabolic disorders. The exact role of mature adipocytes in this phenomenon remains elusive. Here we describe the role of adipose branched-chain amino acid (BCAA) catabolism in this process. We found that adipocyte-specific Crtc2 knockout protected mice from age-associated metabolic decline. Multiomics analysis revealed that BCAA catabolism was impaired in aged visceral adipose tissues, leading to the activation of mechanistic target of rapamycin complex (mTORC1) signaling and the resultant cellular senescence, which was restored by Crtc2 knockout in adipocytes. Using single-cell RNA sequencing analysis, we found that age-associated decline in adipogenic potential of visceral adipose tissues was reinstated by Crtc2 knockout, via the reduction of BCAA-mTORC1 senescence-associated secretory phenotype axis. Collectively, we propose that perturbation of BCAA catabolism by CRTC2 is critical in instigating age-associated remodeling of adipose tissue and the resultant metabolic decline in vivo.

Production of Plant-Derived Japanese Encephalitis Virus Multi-Epitope Peptide in Nicotiana benthamiana and Immunological Response in Mice.

The current production of the Japanese encephalitis virus (JEV) vaccine is based on animal cells, where various risk factors for human health should be resolved. This study used a transient expression system to express the chimeric protein composed of antigenic epitopes from the JEV envelope (E) protein in Nicotiana benthamiana. JEV multi-epitope peptide (MEP) sequences fused with FLAG-tag or 6× His-tag at the C- or N-terminus for the purification were introduced into plant expression vectors and used for transient expression. Among the constructs, vector pSK480, which expresses MEP fused with a FLAG-tag at the C-terminus, showed the highest level of expression and yield in purification. Optimization of transient expression procedures further improved the target protein yield. The purified MEP protein was applied to an ICR mouse and successfully induced an antibody against JEV, which demonstrates the potential of the plant-produced JEV MEP as an alternative vaccine candidate.

Molecular Prevalence of Microsporidia Infection in Patients with Lung Cancer.

Infections are still among the most important causes of morbidity and mortality in patients with lung cancer, which has the highest rate of cancer-related deaths in the world. Microsporidia, which are opportunistic parasitic fungi, primarily localize to the intestine by ingestion but can disseminate to the respiratory tract or can be acquired by spore inhalation. Cancer patients are at higher risk for microsporidia, a life-threatening infection, than the normal population is. We aimed to characterize the prevalence of microsporidia infection for the first time by evaluating the intestinal and respiratory tracts of patients with lung cancer. In this study, we investigated 98 patients with lung cancer and 103 healthy individuals for microsporidia infection and evaluated the clinical findings of patients who were found to be positive. Sputum and stool samples were tested by microscopic examination, in addition to pan-microsporidia and genus-specific polymerase chain reactions. Nine patients with lung cancer had positive results for microsporidia (9.2%), which was significantly higher than the rate in healthy individuals (P = 0.008), and most of them had clinical findings. Among these positive patients, polymerase chain reaction revealed microsporidia in the sputum samples of seven patients, the stool sample of one patient, and both the sputum and stool samples of one patient. Encephalitozoon cuniculi was identified as the predominant pathogen in 87.5% (7/8) of positive sputum samples. Microsporidia infection was significantly associated with advanced stages of cancer. However, in the control group, Encephalitozoon intestinalis was detected in the stool sample of an individual without clinical symptoms. Microsporidia, especially E. cuniculi, should be considered as a cause of respiratory tract infection as well as intestinal infection in cancer patients and should be screened in respiratory samples of these patients when they have pulmonary symptoms.

Flow Field-Flow Fractionation with a Thickness-Tapered Channel.

This study introduces the thickness-tapered channel design for flow field-flow fractionation (FlFFF) for the first time. In this design, the channel thickness linearly decreases along the channel axis such that the flow velocity increases down the channel. Channel thickness is an important variable for controlling retention time and resolution in field-flow fractionation. Especially, in the steric/hyperlayer mode of FlFFF, in which particles (>1 µm) migrate at elevated heights above the channel wall owing to hydrodynamic lift forces, the migration of long-retaining smaller-sized particles can be enhanced in a relatively thin channel or by increasing the migration flow rate; however, an upper size limit that can be resolved is simultaneously sacrificed. A thickness-tapered channel was constructed without a channel spacer by carving the surface of a channel block such that the channel inlet was deeper than the outlet (w = 400 → 200 µm). The performance of a thickness-tapered channel was evaluated using polystyrene standards and compared to that of a channel of uniform thickness (w = 300 µm) with a similar effective channel volume in terms of sample recovery, dynamic size range of separation, and steric transition under different flow rate conditions. The thickness-tapered channel can be an alternative to maintain the resolving power for particles with an upper large-diameter limit, faster separation of particles with a lower limit, and higher elution recovery without implementing the additional field-programming option.

Brain lipidomics: From functional landscape to clinical significance.

Lipids are crucial components of cellular function owing to their role in membrane formation, intercellular signaling, energy storage, and homeostasis maintenance. In the brain, lipid dysregulations have been associated with the etiology and progression of neurodegeneration and other neurological pathologies. Hence, brain lipids are emerging as important potential targets for the early diagnosis and prognosis of neurological diseases. This review aims to highlight the significance and usefulness of lipidomics in diagnosing and treating brain diseases. We explored lipid alterations associated with brain diseases, paying attention to organ-specific characteristics and the functions of brain lipids. As the recent advances in brain lipidomics would have been impossible without advances in analytical techniques, we provide up-to-date information on mass spectrometric approaches and integrative analysis with other omic approaches. Last, we present the potential applications of lipidomics combined with artificial intelligence techniques and interdisciplinary collaborative research for treating brain diseases with clinical heterogeneities.

Optimization for size separation of graphene oxide sheets by flow/hyperlayer field-flow fractionation.

Graphene oxide (GO)-a chemical derivative of graphene with numerous oxygen functional groups on its surface-has attracted considerable interest because of its intriguing properties in relation to those of pristine graphene. In addition to the inherent wide lateral size distribution of GO sheets arising from the typical oxidative exfoliation of graphite, control of the lateral size of GO is critical for desired GO-based applications. Herein, flow/hyperlayer field-flow fractionation (flow/hyperlayer FFF) is optimized to separate GO sheets by lateral dimensions. Optimized fractionation is achieved by investigating the influences of carrier solvent, channel thickness, and flow rate conditions on the steric/hyperlayer separation of GO sheets by flow FFF. Due to the strong hydrodynamic lift forces of extremely thin GO sheets, a thick flow FFF channel (w = 350 µm) and a very low field strength are required to retain the GO sheets within the channel. GO sheets with narrow size fractions are successfully collected from two different graphite sources during flow/hyperlayer FFF runs and are examined to verify the size evolution. Considering the average lateral diameter of the GO fraction calculated on the basis of the assumption of a circular disk shape, the retention of the GO sheets is 2.2-5.0 times faster than that of spherical particles of the same diameter. This study demonstrates that through flow/hyperlayer FFF, the size distribution of GO sheets can be determined and narrow size fractions can be collected (which is desirable for GO-based applications), which are commonly influenced by the GO lateral dimension.

Size Separation of Exosomes and Microvesicles Using Flow Field-Flow Fractionation/Multiangle Light Scattering and Lipidomic Comparison.

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.

Wide-Range Size Fractionation of Graphene Oxide by Flow Field-Flow Fractionation.

Many interesting properties of 2D materials and their assembled structures are strongly dependent on the lateral size and size distribution of 2D materials. Accordingly, effective size separation of polydisperse 2D sheets is critical for desirable applications. Here, we introduce flow field-flow fractionation (FlFFF) for a wide-range size fractionation of graphene oxide (GO) up to 100 µm. Two different separation mechanisms are identified for FlFFF, including normal mode and steric/hyperlayer mode, to size fractionate wide size-distributed GOs while employing a crossflow field for either diffusion or size-controlled migration of GO. Obviously, the 2D GO sheet reveals size separation behavior distinctive from typical spherical particles arising from its innate planar geometry. We also investigate 2D sheet size-dependent mechanical and electrical properties of three different graphene fibers produced from size-fractionated GOs. This FlFFF-based size selection methodology can be used as a generic approach for effective wide-range size separation for 2D materials, including rGO, TMDs, and MXene.

Optimisation of saliva volumes for lipidomic analysis by nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry.

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.

Enhancement of acidic lipid analysis by nanoflow ultrahigh performance liquid chromatography-mass spectrometry.

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.

Optimisation of high-speed lipidome analysis by nanoflow ultrahigh-performance liquid chromatography-tandem mass spectrometry: Application to identify candidate biomarkers for four different cancers.

Lipid analysis is a powerful tool that can elucidate the pathogenic roles of lipids in metabolic diseases, and facilitate the development of potential biomarkers. Lipid analysis by large-scale lipidomics requires a high-speed and high-throughput analytical platform. In the present study, a high-speed analytical method for lipid analysis using nanoflow ultrahigh-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (nUHPLC-ESI-MS/MS) was optimised by investigating the effects of column flow rate, pump flow rate, dwell time, initial binary mobile phase composition, and gradient duration on the separation efficiency of standard lipid mixtures. The minimum gradient time for high-speed lipid separation was determined by examining the time-based separation efficiency and spectral overlap of isobaric lipid species during selected reaction monitoring-based quantification of sphingomyelin and a second isotope of phosphatidylcholine, which differ in molecular weight by only 1 Da. Finally, the optimised nUHPLC-ESI-MS/MS method was applied to analyse 200 plasma samples from patients with liver, gastric, lung, and colorectal cancer to evaluate its performance by measuring previously identified candidate lipid biomarkers. About 73% of the reported marker candidates (6 out of 7 in liver, 5/9 in gastric, 4/6 in lung, and 6/7 in colorectal cancer) could be assigned using the optimised method, supporting its use for high-throughput lipid analysis.

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.

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.

Bifidobacterium bifidum strains synergize with immune checkpoint inhibitors to reduce tumour burden in mice.

The gut microbiome can influence the development of tumours and the efficacy of cancer therapeutics1-5; however, the multi-omics characteristics of antitumour bacterial strains have not been fully elucidated. In this study, we integrated metagenomics, genomics and transcriptomics of bacteria, and analyses of mouse intestinal transcriptome and serum metabolome data to reveal an additional mechanism by which bacteria determine the efficacy of cancer therapeutics. In gut microbiome analyses of 96 samples from patients with non-small-cell lung cancer, Bifidobacterium bifidum was abundant in patients responsive to therapy. However, when we treated syngeneic mouse tumours with commercial strains of B. bifidum to establish relevance for potential therapeutic uses, only specific B. bifidum strains reduced tumour burden synergistically with PD-1 blockade or oxaliplatin treatment by eliciting an antitumour host immune response. In mice, these strains induced tuning of the immunological background by potentiating the production of interferon-γ, probably through the enhanced biosynthesis of immune-stimulating molecules and metabolites.

Lipidomic signatures of post-hepatectomy liver failure using porcine hepatectomy models.

BACKGROUND: Clinical diagnosis of post-hepatectomy liver failure (PHLF) can only be made on or after the 5th postoperative day. Biomarker for early diagnosis is considered as a critical unmet need. METHODS: Twenty domestic female crossbreed (Yorkshire-landrace and duroc) pigs underwent sham operation (n=6), 70% (n=7) and 90% (n=7) partial hepatectomy (PH). A comprehensive lipidomic analysis was conducted using sera collected at pre-operation (PO), 14, 30, and 48 h after PH using nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry. RESULTS: Of the 184 quantified lipids, 14 lipids showed significant differences between the two resection groups starting at 30 h after surgery. Four phosphatidylcholine (PC) plasmalogen species (P-16:0/16:0, P-18:0/18:2, P-18:0/20:4, and P-18:0/22:6) and PC 32:2 significantly increased in the 90% PH group while these returned to PO level after 30 h in the 70% PH group, presumably implying the failure markers. In contrast, eight triacylglycerol (TG) species (40:0, 42:1, 42:0, 44:1, 44:2, 46:1, 46:2, and 48:3) and sphingomyelin d18:1/20:0 showed an opposite trend, wherein they significantly decreased in the 90% PH group while these in the 70% PH group were abruptly increased until 30 h but returned to near PO levels at 48 h, implying the recovery markers. Same trends could also be observed in the level of whole lipid classes of PC plasmalogens and TGs, in addition to selected individual lipid species. CONCLUSIONS: Characteristic lipidomic signatures of PHLF could be identified using large animal models. These candidates have a potential to serve as a tool for early diagnosis and may open new paths to the study to overcome PHLF.

Exercise-induced recovery of plasma lipids perturbed by ageing with nanoflow UHPLC-ESI-MS/MS.

Daily physical exercise is an essential part of life and is required for remaining healthy; it enhances therapeutic efficacy in the elderly and prevents age-related diseases associated with lipid profile alterations, such as cardiovascular disease, diabetes mellitus, and dementia. To more efficiently analyse the lipid profiles and unveil the effect of exercise in aged mice, we optimized our study by examining the effects of using ionization modifiers in the mobile phase and in-source fragmentation of lysophospholipids on the simultaneous analysis of fatty acids (FAs) including hydroxyl fatty acids, glycerophospholipids, sphingolipids, and glycerolipids using nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry. We applied the optimization to investigate the lipidomic plasma alterations in young (7 weeks old) and aged (84 weeks old) mice (C57BL/6) subjected to treadmill exercise. Of the 390 identified lipid species, 159 were quantified to investigate ageing-related lipid species responsive to physical exercise. In particular, circulating lysophosphatidylcholine and lysophosphatidylethanolamine levels showed a significant decrease, and lysophosphatidic acid showed a simultaneous increase with ageing. The saturated FA (16:0 and 18:0) increased with ageing while the unsaturated FA 22:6 decreased. Dihydroxy fatty acid (18:1_2OH) showed an exercise-induced recovery against ageing. It is notable that the levels of five triacylglycerol species significantly increased by as much as threefold with ageing, but their levels largely recovered to those observed in the young mice after exercise. These findings can help understand the influence of ageing on lipid perturbation and the role of physical exercise on lipidomic recovery in response to ageing-associated loss of physical status. Graphical abstract.

Evaluation of exosome separation from human serum by frit-inlet asymmetrical flow field-flow fractionation and multiangle light scattering.

Exosomes are extracellular vesicles that mediate intercellular communication, immune response, and tumour metastasis. However, exosome isolation from the blood is complicated because their size and density are similar to those of blood lipoproteins. Here, we employed field programming frit-inlet asymmetrical flow field-flow fractionation (FIAF4) coupled with multiangle light scattering (MALS) for the effective separation of exosomes from free unbound proteins and lipoproteins present in serum samples using different pre-treatment methods, namely, a commercial exosome isolation kit, ultracentrifugation (UC), and a simple centrifugation followed by ultrafiltration (UF). Sizes of the eluted exosomes, as calculated by MALS signals, approximated well with the results of batch dynamic light scattering of the collected fractions and with the sizes of polystyrene particles. Exosome separation from lipoproteins was validated by western blotting with several markers of exosomes and lipoproteins, followed by proteomic analysis using nanoflow ultrahigh-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry. UC requires relatively large amounts of serum samples (at least 2 mL) but is more efficient at removing lipoproteins. The UF method with a centrifugal concentrator (300 kDa) was found to be more effective in retrieving exosomes with low serum volumes (50 µL). Altogether, this study demonstrates the application of field programming FIAF4 for the isolation/purification of exosomes from proteins and lipoproteins in the serum.