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Urinary exosome isolation using multiple cycles of centrifugation
Iqbal Jalaludin,김정권 한국분석과학회 2021 학술대회논문집 Vol.2021 No.11
Exosomes have great potential for non-invasive diagnostics and therapeutics because of their capacity in facilitating communication between cells and drug delivery. Exosomes are produced in cells and discharged into the extracellular milieu as they circulate throughout the system. Hence, they can be found in biofluids such as blood and urine. Ultracentrifugation is a standard method for isolating exosomes that use high-speed centrifugation, i.e. 100,000×g, to pellet exosomes. However, the exosome integrity can be compromised by repetitive high-speed centrifugation, which damages the vesicles. Our present study employed low-speed centrifugation, i.e. 40,000×g, to isolate exosomes from urine samples. The method required four cycles of centrifugation to remove non-exosomal proteins based on the gel electrophoresis results. Exosomal proteins like CD63 and TSG101 corresponded to the antibodies in Western blotting, confirming the existence of exosomes. Dynamic light scattering profiles suggested that the average diameter of the isolated pellets was 50 to 200 nm, which is the size range of exosomes. All characterization methods indicate that the urinary exosome isolation using lower speed centrifugation was feasible. Further research with a larger group of subjects is needed to assess the reproducibility of the method.
MALDI-MS: A Powerful but Underutilized Mass Spectrometric Technique for Exosome Research
Iqbal Jalaludin,David M. Lubman,김정권 사단법인 한국질량분석학회 2021 Mass spectrometry letters Vol.12 No.3
Exosomes have gained the attention of the scientific community because of their role in facilitating intercellular communi- cation, which is critical in disease monitoring and drug delivery research. Exosome research has grown significantly in recent decades, with a focus on the development of various technologies for isolating and characterizing exosomes. Among these efforts is the use of matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS), which offers high-throughput direct analysis while also being cost and time effective. MALDI is used less frequently in exosome research than electrospray ionization due to the diverse popu- lation of extracellular vesicles and the impurity of isolated products, both of which necessitate chromatographic separation prior to MS analysis. However, MALDI-MS is a more appropriate instrument for the analytical approach to patient therapy, given it allows for fast and label-free analysis. There is a huge drive to explore MALDI-MS in exosome research because the technology holds great potential, most notably in biomarker discovery. With methods such as fingerprint analysis, OMICs profiling, and statistical analysis, the search for biomarkers could be much more efficient. In this review, we highlight the potential of MALDI-MS as a tool for investigating exo- somes and some of the possible strategies that can be implemented based on prior research.
MALDI-MS analysis of disaccharide isomers using graphene oxide matrix with different cation agent
권도경,Iqbal Jalaludin,김정권 한국분석과학회 2021 학술대회논문집 Vol.2021 No.11
Disaccharides consist of two monosaccharides joined by a glycosidic linkage. The differences in disaccharide isomers are related to their constituent monosaccharides, anomeric configuration, and type of glycosidic linkage. In this study, seven disaccharide isomers (cellobiose, melibiose, maltose, lactose, sucrose, gentiobiose, and isomaltose) were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using a graphene oxide matrix with different cationization agents (Na⁺, K⁺, and Ag⁺). More disaccharide isomers fragmentation occurred when Na⁺ was used. The seven fragmentation peaks were detected when Na⁺ was used, while two fragmentation peaks detected when K⁺ or Ag⁺ was used. An adduct form of disaccharide isomers with sodium, potassium, or silver ion was detected at m/z 365, m/z 381, or m/z 449, respectively. To identifiy disaccharide isomers, tandem mass spectrometry (MS/MS) is used for all the seven isomers. The current results will be beneficial for differenciating disaccharide isomers using MALDI-MS.
정신희,Jalaludin Iqbal,김정권 한국분석과학회 2021 학술대회논문집 Vol.2021 No.11
Top-down proteomics is a powerful strategy to obtain the complete protein sequence and to locate and characterize post-translational modifications. Furthermore, the time-consuming protein digestion required for bottom-up methods is eliminated. In top-down proteomics, intact proteins are analyzed by a mass spectrometer without prior digestion of the proteins to peptides. Polyacrylamide gel electrophoresis, which is an easy and cost-effective way to prefractionate protein mixtures into individual proteins depending on their size, is a technique widely used in biochemical experiments and has the potential to be effective in sample fractionation for top-down mass spectrometry analysis. However, it is difficult to extract prefractionated intact proteins from the gel. In general, expensive specialized laboratory equipment is required to extract intact proteins from the gel. In this study, we used a new extraction solution mixture of 100 mM triethylamine hydrogen sulfate and 0.1 % (v/w) sodium dodecyl sulfate and a syringe filter for a successful extraction of proteins from the gel. One-dimensional gel separation was first applied to bovine serum albumin, which was extracted by using the new extraction solution mixture and the syringe filter. One-dimensional gel separation was applied again to the extracted albumin to check the efficiency of the first extraction. The intensity of protein bands was quantified by image J. Quantitative analysis showed that the proteins were efficiently extracted from the gel.