A guide to mass spectrometric analysis of extracellular vesicle proteins for biomarker discovery.

Exosomes (small extracellular vesicles) in living organisms play an important role in processes such as cell proliferation or intercellular communication. Recently, exosomes have been extensively investigated for biomarker discoveries for various diseases. An important aspect of exosome analysis involves the development of enrichment methods that have been introduced for successful isolation of exosomes. These methods include ultracentrifugation, size exclusion chromatography, polyethylene glycol-based precipitation, immunoaffinity-based enrichment, ultrafiltration, and asymmetric flow field-flow fractionation among others. To confirm the presence of exosomes, various characterization methods have been utilized such as Western blot analysis, atomic force microscopy, electron microscopy, optical methods, zeta potential, visual inspection, and mass spectrometry. Recent advances in high-resolution separations, high-performance mass spectrometry and comprehensive proteome databases have all contributed to the successful analysis of exosomes from patient samples. Herein we review various exosome enrichment methods, characterization methods, and recent trends of exosome investigations using mass spectrometry-based approaches for biomarker discovery.

Sterilization efficacy of a homemade UV lamp system on ceramic and porcelain tiles.

Ultraviolet (UV) sterilization of Bacillus atrophaeus spores attached to eight types of tiles, consisting of combinations of ceramic and porcelain, white and black, and matte and glossy surfaces, was investigated using a homemade UV lamp system with different irradiation times (10 s, 30 s, and 60 s) and UV lamp-to-tile distances (32 mm, 76 mm, and 120 mm). The results demonstrated a reduction in colony numbers with increasing irradiation time and decreasing lamp-to-tile distance, with nearly complete sterilization observed for a 120 mm lamp-to-tile distance with 60 s UV irradiation and for a 32 mm lamp-to-tile distance with 10 s UV irradiation. Specifically, superior UV sterilization efficacy was observed on porcelain compared to ceramic tiles, on white compared to black tiles, and on matte compared to glossy tiles, consistent with the reflectance trend. In conclusion, among the tested tile surfaces, the white matte porcelain tile exhibited the most efficient UV sterilization, attributed to its highest UV reflectance.

Matrix-assisted laser desorption/ionization-Fourier-transform ion cyclotron resonance-mass spectrometry analysis of exosomal lipids from human serum.

RATIONALE: Exosomes contain biomarkers such as proteins and lipids that help in understanding normal physiology and diseases. Lipids, in particular, are infrequently studied using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) for biomarker discovery. In this study, MALDI was equipped with a high-resolution MS to investigate exosomal lipids from human serum. METHODS: Exosomal lipids were profiled using MALDI with Fourier-transform ion cyclotron resonance (FTICR)-MS. Four matrices (i.e., α-cyano-4-hydroxycinnamic acid [CHCA], 2,5-dihydroxybenzoic acid, sinapinic acid, and graphene oxide [GO]) and three sample preparation methods (i.e., dried droplet, thin layer, and two layer) were compared for the number of lipid species detected and the relative abundance of each lipid from human serum and human serum exosomes. RESULTS: In sum, 172 and 89 lipid species were identified from human serum and human serum exosomes, respectively, using all the methods. The highest number of exosome lipid species, 69, was detected using the CHCA matrix, whereas only 8 exosome lipid species were identified using the GO matrix. Among the identified lipid species, phosphatidylcholine was identified most frequently, probably due to the use of a positive ion mode. CONCLUSIONS: Exosomes and human serum showed comparable lipid profiles as determined using MALDI-FTICR-MS. These findings provide a new perspective on exosomal lipidomics analysis and may serve as a foundation for future lipidomics-based biomarker research using MALDI-FTICR-MS.

Detection of multiply charged protein ions using matrix-assisted laser desorption/ionization mass spectrometry and a force-dried droplet method with a 2-nitrophloroglucinol matrix.

Conventional dried droplet (DD) methods show poor reproducibility in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) due to the frequent induction of a heterogeneous sample distribution. Recently, a forced dried droplet (FDD) sample preparation method was introduced to form homogeneous samples; this method improves the reproducibility of MALDI-MS analysis and generates highly multiply charged ions compared to DD methods. The FDD method utilizes secondary nucleation to generate a homogeneous sample distribution by applying an external force such as fluid shear stress by stirring the sample using a micropipette tip. In this study, a 2-nitrophloroglucinol (2-NPG) matrix was used for the DD and FDD sample preparation methods, and the charge state and homogeneity were compared by detecting multiply charged ions of proteins including cytochrome c, myoglobin, and immunoglobulin G (IgG) and measuring the relative standard deviation (RSD). FDD with a 2-NPG matrix produced a more homogeneous sample distribution and higher charge state ions than the DD method. FDD with a 2-NPG matrix was applied in MALDI-MS analysis of IgG fragments obtained from sequential reduction of IgG. In addition, FDD with intentional scratching of the MALDI plate by rotating a micropipette tip was found to provide similar or better reproducibility, higher charge state ions, and more uniformly distributed sample morphology compared to FDD without scratching.

Sterilization effects of UV laser irradiation on Bacillus atrophaeus spore viability, structure, and proteins.

Bacillus spores are highly resistant to toxic chemicals and extreme environments. Because some Bacillus species threaten public health, spore inactivation techniques have been intensively investigated. We exposed Bacillus atrophaeus spores to a 266 nm Nd:YVO4 laser at a laser power of 1 W and various numbers of scans. As a result, the UV laser reduced the viability of Bacillus atrophaeus spores. Although the outer coat of spores remained intact after UV laser irradiation of 720 scans, damage inside the spores was observed. Spore proteins were identified by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry during the course of UV laser irradiation. Photochemical and photothermal processes are believed to be involved in the UV laser sterilization of Bacillus spores. Our findings suggest that a UV laser is capable of sterilizing Bacillus atrophaeus spores.

Platelet Factor 4 as a Novel Exosome Marker in MALDI-MS Analysis of Exosomes from Human Serum.

Exosomes are nanosized vesicles commonly found in biological fluids as a result of a secretion process involving endosomes and multivesicular bodies. The isolation and analysis of exosomes can be useful for noninvasive clinical diagnosis of a variety of human diseases. We investigated the utility of analyzing exosomal proteins, using matrix-assisted laser desorption/ionization combined with Fourier-transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS), as a means of determining the presence of exosomes. MALDI-FTICR-MS analyses of exosomes enriched from human serum via centrifugation in a mass range of m/z 1000-20 000 yielded a distinctive protein around m/z 7766. The high mass accuracy and resolution of MALDI-FTICR-MS allowed for reliable comparisons against a protein database, through which the protein was identified as platelet factor 4 (PLF4), whose singly charged protein peak has an elemental composition of C341H577N96O101S4+, with a theoretical most abundant isotopic peak at m/z 7765.194 and a theoretical average peak at m/z 7766. The MALDI-TOF MS analysis of exosomes from the serum of 27 patients with different states of liver diseases provided the most abundant PLF4 peak for each mass spectrum, along with several additional minor peaks. In conclusion, MALDI-MS is suitable as an alternative exosome detection method, serving as a valuable confirmation tool, greatly decreasing the time and workload associated with exosome identification.

Effects of incubation temperature and acetonitrile amount on microwave-assisted tryptic digestion of proteins.

The effects of incubation temperature and acetonitrile (ACN) amount on microwave-assisted tryptic digestion of horse skeletal muscle myoglobin (MYG) and bovine serum albumin (BSA) were investigated. Microwave-assisted tryptic digestion was performed on BSA or MYG solutions containing different amounts (0, 10, and 20%) of ACN for different times (10, 20, 30, 40, and 50 min) at different temperatures (25, 37, and 55 °C). Conventional overnight tryptic digestion was also conducted with gentle mixing at 37 °C for 16 h. Digested samples were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. Similar sequence coverage (SC) values were obtained under most conditions except when the protein sample solutions were digested with 20% ACN at 55 °C, which provided the lowest SC for both MYG and BSA for all investigated digestion times. Considering the missed cleavage (MC) ratios for 50-min microwave-assisted digestion, the highest MC ratio, (i.e., lowest trypsin activity) was observed for the digestion condition of 20% ACN at 55 °C for both proteins, while the lowest MC ratio was observed with 0% ACN at 25 °C for MYG and with 0% ACN at 55 °C for BSA. Conventional overnight tryptic digestion at 37 °C provided more completely cleaved peptides than 50-min microwave-assisted tryptic digestion at the same temperature.

Microwave-Assisted Protein Digestion in a Plate Well for Facile Sampling and Rapid Digestion.

Protein digestion is one of the most important processes in proteomic analysis. Here, we report microwave-assisted protein digestion in a plate well, which allows for facile sampling as well as rapid protein digestion based on the combination of highly stable enzyme immobilization and 3D printing technologies. Trypsin (TR) was immobilized on polystyrene-based nanofibers via an enzyme coating (EC) approach. The EC with stabilized TR activity was assembled with the 3D-printed structure in the plate well (EC/3D), which provides two separated compartments for the solution sampling and the TR-catalyzed protein digestion, respectively. EC/3D can effectively prevent the interference of sampling by accommodating EC in the separated compartment from the sampling hole in the middle. EC/3D in the plate well maintained its protein digestion performance under shaking over 160 days. Microwave irradiation enabled the digestion of bovine serum albumin within 10 min, generating the MALDI-TOF MS results of 75.0% sequence coverage and 61 identified peptides. EC/3D maintained its protein digestion performance under microwave irradiation after 30 times of recycled uses. EC/3D in the plate well has demonstrated its potential as a robust and facile tool for the development of an automated protein digestion platform. The combination of stable immobilized enzymes and 3D-printed structures can be potentially utilized not only for the protein digestion, but also for many other enzyme applications, including bioconversion and biosensors.

Prevalence rate and clinical characteristics of esophageal ectopic sebaceous glands in asymptomatic health screen examinees.

Ectopic sebaceous glands in the esophagus have rarely been reported and, thus, represent an obscure medical condition. The aim of this study is to identify the prevalence rate and clinical characteristics of this lesion in an asymptomatic population. We prospectively enrolled health screen examinees who underwent esophagogastroduodenoscopy for gastric cancer screening. An esophageal biopsy was performed in the cases in which esophageal ectopic sebaceous glands were suspected. The general characteristics of the examinees were analyzed based on their medical records. A total of 9989 examinees were enrolled, and five examinees were diagnosed with esophageal ectopic sebaceous glands between December 2012 and June 2014. The endoscopic findings of the esophageal ectopic sebaceous glands indicated multiple yellowish patches or papules, which varied in size. The histopathological findings indicated several lobulated sebaceous glands in the squamous epithelium with inflammatory infiltration. The follow-up endoscopic findings indicated that there was no grossly discernible change. In conclusion, esophageal ectopic sebaceous glands are present in 0.05% of asymptomatic subjects. This lesion is thought to be benign and is not related to clinical symptoms. Therefore, esophageal ectopic sebaceous glands do not require further treatment or follow-up, which makes endoscopists free from active efforts for differential diagnosis with other malignant diseases.

Exosome enrichment of human serum using multiple cycles of centrifugation.

In this work, we compared the use of repeated cycles of centrifugation at conventional speeds for enrichment of exosomes from human serum compared to the use of ultracentrifugation (UC). After removal of cells and cell debris, a speed of 110 000 × g or 40 000 × g was used for the UC or centrifugation enrichment process, respectively. The enriched exosomes were analyzed using the bicinchoninic acid assay, 1D gel separation, transmission electron microscopy, Western blotting, and high-resolution LC-MS/MS analysis. It was found that a five-cycle repetition of UC or centrifugation is necessary for successful removal of nonexosomal proteins in the enrichment of exosomes from human serum. More significantly, 5× centrifugation enrichment was found to provide similar or better performance than 5× UC enrichment in terms of enriched exosome protein amount, Western blot band intensity for detection of CD-63, and numbers of identified exosome-related proteins and cluster of differentiation (CD) proteins. A total of 478 proteins were identified in the LC-MS/MS analyses of exosome proteins obtained from 5× UCs and 5× centrifugations including many important CD membrane proteins. The presence of previously reported exosome-related proteins including key exosome protein markers demonstrates the utility of this method for analysis of proteins in human serum.

Analysis of Self-Assembled Micelle Inhibitory RNA (SAMiRNA) Drug Using Ion-Pairing Reversed-Phase Liquid Chromatography Combined with Mass Spectrometry.

Small interfering RNA (siRNA) is known for its ability to silence the expression of specific genes, demonstrating its promising potential as a therapeutic approach. Self-assembled micelle inhibitory RNA (SAMiRNA) is an oligonucleotide duplex developed to overcome the in vivo delivery limitations of siRNA. SAMiRNA has hydrophilic and hydrophobic groups at both ends of a sense strand, forming a spherical nanostructure that enhances the in vivo delivery efficiency. Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the most commonly used method for the analysis of oligonucleotides. Since SAMiRNA is heavily chemically modified, the behavior of SAMiRNA in IP-RPLC combined with mass spectrometry (MS) is anticipated to differ from that of the conventional siRNA drug. The current investigation using IP-RPLC-MS revealed that a distinct duplex peak along with two minor separate strands of antisense and sense was observed at column temperatures below 35 °C in the IP-RPLC system with a 100 mM ammonium bicarbonate buffer system. At column temperatures higher than 35 °C, however, two fully denatured single strands were observed. The mass spectrum from the chromatographic peak of the SAMiRNA duplex contained signals from the duplex, the antisense, and the sense, probably due to duplex denaturation during the MS ionization process. The current comprehensive analysis results will make a substantial contribution to the future application of IP-RPLC-MS in the analysis of SAMiRNA.

Ultraviolet Light-Assisted Decontamination of Chemical Warfare Agent Simulant 2-Chloroethyl Phenyl Sulfide on Metal-Loaded TiO2 /Ti Surfaces.

The application of ultraviolet (UV) light for the decontamination of chemical warfare agents (CWAs) has gained recognition as an effective method, especially for treating hard-to-reach areas where wet chemical methods are impractical. In this study, TiO2 /Ti was employed as a model catalyst, which was contaminated with 2-chloroethyl phenyl sulfide (CEPS), and subjected to photocatalytic decontamination using both UVB and UVC light. Additionally, photocatalytic decontamination efficiency by introducing Au, Pt, and Cu onto the TiO2 /Ti surface was explored. During the photodecomposition process under UVC light, at least eight distinct secondary byproducts were identified. It was observed that the introduction of overlayer metals did not significantly enhance the photodecomposition under UVC light instead overlaid Au exhibited substantially improved activity under UVB light. Whereas, photodecomposition process under UVB light, only five secondary products were detected, including novel compounds with sulfoxide and sulfone functional groups. This novel study offers valuable insights into the generation of secondary products and sheds light on the roles of overlayer metals and photon wavelength in the photodecontamination process of CWA.

Potential applications of mesenchymal stem cells and their derived exosomes in regenerative medicine.

INTRODUCTION: Regenerative medicine involves the replacement of damaged cells, tissues, or organs to restore normal function. Mesenchymal stem cells (MSCs) and exosomes secreted by MSCs have unique advantages that make them a suitable candidate in the field of regenerative medicine. AREAS COVERED: This article provides a comprehensive overview of regenerative medicine, focusing on the use of MSCs and their exosomes as potential therapies for replacing damaged cells, tissues, or organs. This article discusses the distinct advantages of both MSCs and their secreted exosomes, including their immunomodulatory effects, lack of immunogenicity, and recruitment to damaged areas. While both MSCs and exosomes have these advantages, MSCs also have the unique ability to self-renew and differentiate. This article also assesses the current challenges associated with the application of MSCs and their secreted exosomes in therapy. We have reviewed proposed solutions for improving MSC or exosome therapy, including ex-vivo preconditioning strategies, genetic modification, and encapsulation. Literature search was conducted using Google Scholar and PubMed databases. EXPERT OPINION: Providing insight into the future development of MSC and exosome-based therapies and to encourage the scientific community to focus on the identified gaps, develop appropriate guidelines, and enhance the clinical application of these therapies.

Identification of geographical origins of soybean pastes using headspace gas chromatography-mass spectrometry by selecting sample-descriptive components with an Incremental Association Markov Blanket.

The identification of geographical origins of soybean pastes using headspace gas chromatography-mass spectrometry was attempted in this study. Since soybean paste was odor-rich, 36 components were identified in the imported and domestic soybean samples. t-Test, variable importance in projection (VIP), and Incremental Association Markov Blanket (IAMB) were employed to select proper components that could effectively discriminate the two sample groups. The discrimination accuracies were below 87.3 % when all 36 components were fed for either LDA, k-NN, or SVM. When the five t-test-selected components or six VIP score-selected components were employed, the accuracies improved to 95.2-96.2 %. The IAMB selected three different components were 3-methylbutanal, 4-methylnonane, and 2,3-pentanedione, and the correlations among their peak areas were not significant. This suggests that these three components were independently relevant for the discrimination. The accuracy obtained using these three components was superior, 97.7 %, as undescriptive and/or redundant components for the discrimination were excluded.

Accurate determination of 11 representative phthalates and di(2-ethylhexyl) terephthalate in polyvinyl chloride using isotope dilution-gas chromatography/mass spectrometry.

Phthalates are mainly used as plasticizers in polyvinyl chloride (PVC). However, prolonged exposure to phthalates poses considerable risks to human health. Consequently, the utilization of phthalates in consumer products is subject to regulations, with a defined threshold of 0.1 %. In this study, we developed an accurate and simultaneous method for determination of 11 representative phthalates and a non-phthalate plasticizer (di(2-ethylhexyl) terephthalate, DEHT) in PVC as a higher-order reference method. Homogeneously prepared PVC samples, each containing approximately 0.1 % of the target plasticizer compounds, were analyzed using gas chromatography-mass spectrometry (GC-MS) with deuterium-labeled phthalates and DEHT. The developed method could effectively separate and quantify all target plasticizers without interference with each other and potential overlap between the isomeric forms of phthalates, di-isodecyl phthalate, and di-isononyl phthalate. The developed method has high-order metrological quality, exhibiting exceptional selectivity, accuracy, repeatability (≤ 2.17 %), reproducibility (≤ 2.16 %), and relative expanded uncertainty (≤ 5.6 %). This analytical method is thus suitable for accurately assessing the target plasticizer levels in PVC products for ensuring compliance with the established 0.1 % threshold. This method was successfully applied to quantify twelve distinct plasticizers in PVC products obtained from the Korean market, validating its effectiveness and reliability in real-world scenarios.

Size-exclusion chromatography for the characterization of urinary extracellular vesicles.

In recent years, extracellular vesicles (EVs) have gained attention for their potential as biomarkers for the early diagnosis and treatment of various diseases. Traditionally, EV isolation has relied exclusively on ultracentrifugation. However, alternative enrichment methods such as size-exclusion chromatography (SEC) and polyethylene glycol-based precipitation have been introduced. This study utilized SEC as a characterization tool to assess the efficiency of EV isolation. Urinary EVs isolated from human urine using centrifugation (40,000 × g) were analyzed using an SEC column with a pore size of 1000 Å, an inner diameter of 7.8 mm, and a length of 300 mm. The EVs were detected sequentially using UV (280 nm) and fluorescence (λex/em = 550 nm/565 nm); the EVs were observed at approximately 6 min, while the proteins were observed at approximately 12 min. The repeated centrifugation enrichment steps resulted in an increase in EV peaks and a decrease in protein peaks. SEC analysis of the enriched EV samples confirmed that a four-cycle repetition of centrifugation is necessary for successful EV enrichment and removal of non-EV proteins from 40 mL of human urine.

Evaluation of the effect of dimethyl fumarate on human bone marrow-derived mesenchymal stem cells using bottom-up proteomics.

Mesenchymal stem cells (MSCs) have potential as a viable treatment option in the field of regenerative medicine, but MSC-based therapy needs to be more efficient. Preconditioning is a method to improve MSC-based therapy, and dimethyl fumarate (DMF) - an agent that can enhance the antioxidative capacity of cells - can be considered for preconditioning of MSCs. In this study, we treated bone marrow-derived MSCs with DMF and evaluated their proteome using bottom-up proteomics. The MSCs were exposed to 10 µM DMF for 24 h, followed by lysis with an SDS solution, digestion with trypsin using an s-trap column, and analysis using nanoLC-MS/MS, which identified 2262 proteins with confidence. Bioinformatic analysis of the identified proteins revealed 47 upregulated proteins and 81 downregulated proteins upon DMF treatment. Pathway enrichment analysis suggested a possible decrease in autophagy and a decrease in the activity of the TCA cycle, while indicating a potential increase in proliferation and antioxidant activity in DMF-treated MSCs compared to untreated MSCs. Our findings suggest that DMF can enhance the proliferation of MSCs and increase their stability, and that preconditioning could improve the therapeutic efficacy of MSCs for the treatment of regenerative diseases.

Analysis of cancer cell lipids using matrix-assisted laser desorption/ionization 15-T Fourier transform ion cyclotron resonance mass spectrometry.

A combination of methodologies using the extremely high mass accuracy and resolution of 15-T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) was introduced for the identification of intact cancer cell phospholipids. Lipids from a malignant glioma cell line were initially analyzed at a resolution of >200,000 and identified by setting the mass tolerance to ±1 mDa using matrix-assisted laser desorption/ionization (MALDI) 15-T FT-ICR MS in positive ion mode. In most cases, a database search of potential lipid candidates using the exact masses of the lipids yielded only one possible chemical composition. Extremely high mass accuracy (<0.1 ppm) was then attained by using previously identified lipids as internal standards. This, combined with an extremely high resolution (>800,000), yielded well-resolved isotopic fine structures allowing for the identification of lipids by MALDI 15-T FT-ICR MS without using tandem mass spectrometric (MS/MS) analysis. Using this method, a total of 38 unique lipids were successfully identified.