Separation-Assisted Analysis of Large Biological Complexes

There are multiple challenges and limitations in the current separation methods for large biological complexes, including insufficient resolution, time-consuming pre-treatment processes and downstream analysis, potential analyte-column interactions leading to denaturation or adsorption, and the need for improving efficiency in separation and characterization when dealing with large complexes with small molecules simultaneously. In this dissertation, I aim to develop improved solutions and strategies to overcome these challenges, focusing on two powerful analytical separation techniques: Asymmetrical Flow Field-Flow Fractionation (AF4) and Capillary Electrophoresis (CE). AF4, a separation technique without a stationary phase, has proven valuable in separating large analytes with sizes ranging from nanometers to micrometers over the past three decades, offering an effective alternative to Size Exclusion Chromatography (SEC). Meanwhile, CE has matured into a robust analytical separation technique, featuring advantages like minimal sample consumption, rapid analysis, high resolution, and efficiency.In Chapter 2, the offline coupling of AF4 and CE is detailed for extracellular vesicle (EV) analysis. EVs, as important mediators of cell-to-cell communication capturing great research interests to study their functions and explore their utility in biomedical practices, pose challenges in purification due to their heterogeneity and low abundance in biological samples. AF4 provides rapid size-based separation, while CE resolves EVs from contaminants with similar sizes eluted from AF4. The coupled AF4-CE system demonstrates efficiency in monitoring EV secretion from cells in the cell culture media, and detecting serum EVs, offering rapid quantification in biological samples. Chapter 3 introduces the coupling system of AF4 with Large Volume Sample Stacking/CE (LVSS/CE) to obtain high-purity EVs for downstream protein profiling. This two-dimensional separation method first separates EVs from smaller serum proteins by AF4 and then resolves EVs from non-vesicular matrix components by CE following LVSS. The approach enhances EV isolation and collection from LVSS/CE, enabling EV immuno-fluorescence labeling and permitting the identification of EV proteins through proteomic analysis. Chapter 4 presents an online separation and characterization platform for mRNA and mRNA-loaded lipid nanoparticles (LNPs) using AF4 with multi-detectors (MD-AF4). LNPs, promising in drug delivery, present challenges in characterization. The MD-AF4 method achieves exceptional resolution between mRNA-LNPs and mRNAs, offering comprehensive and multi-attribute particle characterizations within a single injection, including size distribution, batch-to-batch variability, shape factor and encapsulation efficiency. The platform also proves potential for monitoring LNP stability under various stress conditions. This dissertation highlights the significance of AF4 and CE in advancing the analysis of large biological complexes. The novel methodologies introduced herein hold promise for consolidating our understanding of complex biological matrices, and advancing biomedical applications.

Recent (2018-2020) development in capillary electrophoresis.

Development of new capillary electrophoresis (CE) methodology and instrumentation, as well as application of CE to solve new problems, remains an active research area because of the attractive features of CE compared to other separation techniques. In this review, we focus on the representative works about sample preconcentration, separation media or capillary coating development, detector construction, and multidimensional separation in CE, which are judiciously selected from the papers published in 2018-2020

Recent (2018-2020) development in capillary electrophoresis.

Development of new capillary electrophoresis (CE) methodology and instrumentation, as well as application of CE to solve new problems, remains an active research area because of the attractive features of CE compared to other separation techniques. In this review, we focus on the representative works about sample preconcentration, separation media or capillary coating development, detector construction, and multidimensional separation in CE, which are judiciously selected from the papers published in 2018-2020

HERMITIAN REPRESENTATIONS OF THE EXTENDED AFFINE LIE ALGEBRA ˜ gl 2(Cq)

Dedicated to Professor Kyoji Saito on the occasion of his sixtieth birthda

Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles.

Extracellular vesicles (EVs) play important roles in cell-to-cell communications and carry high potential as markers targeted in disease diagnosis, prognosis, and therapeutic development. The main obstacles to EV study are their high heterogeneity; low amounts present in samples; and physical similarity to the abundant, interfering matrix components. Multiple rounds of separation and purification are often needed prior to EV characterization and function assessment. Herein, we report the offline coupling of asymmetrical flow field-flow fractionation (AF4) and capillary electrophoresis (CE) for EV analysis. While AF4 provides gentle and fast EV separation by size, CE resolves EVs from contaminants with similar sizes but different surface charges. Employing Western Blotting, ELISA, and SEM, we confirmed that intact EVs were eluted within a stable time window under the optimal AF4 and CE conditions. We also proved that EVs could be resolved from free proteins and high-density lipoproteins by AF4 and be further separated from the low-density lipoproteins co-eluted in AF4. The effectiveness of the coupled AF4-CE system in EV analysis was demonstrated by monitoring the changes in EV secretion from cells and by direct injection of human serum and detection of serum EVs. We believe that coupling AF4 and CE can provide rapid EV quantification in biological samples with much reduced matrix interference and be valuable for the study of total EVs and EV subpopulations produced by cells or present in clinical samples

Unveiling the Vital Role of Long Non-Coding RNAs in Cardiac Oxidative Stress, Cell Death, and Fibrosis in Diabetic Cardiomyopathy

Diabetes mellitus is a burdensome public health problem. Diabetic cardiomyopathy (DCM) is a major cause of mortality and morbidity in diabetes patients. The pathogenesis of DCM is multifactorial and involves metabolic abnormalities, the accumulation of advanced glycation end products, myocardial cell death, oxidative stress, inflammation, microangiopathy, and cardiac fibrosis. Evidence suggests that various types of cardiomyocyte death act simultaneously as terminal pathways in DCM. Long non-coding RNAs (lncRNAs) are a class of RNA transcripts with lengths greater than 200 nucleotides and no apparent coding potential. Emerging studies have shown the critical role of lncRNAs in the pathogenesis of DCM, along with the development of molecular biology technologies. Therefore, we summarize specific lncRNAs that mainly regulate multiple modes of cardiomyopathy death, oxidative stress, and cardiac fibrosis and provide valuable insights into diagnostic and therapeutic biomarkers and strategies for DCM

Enhancing Extracellular Vesicle Analysis by Integration of Large-Volume Sample Stacking in Capillary Electrophoresis with Asymmetrical Flow Field-Flow Fractionation

Extracellular vesicles (EVs) play important roles in cell–cell communication and pathological development. Cargo profiling for the EVs present in clinical specimens can provide valuable insights into their functions and help discover effective EV-based markers for diagnostic and therapeutic purposes. However, the highly abundant and complex matrix components pose significant challenges for specific identification of low-abundance EV cargos. Herein, we combine asymmetrical flow field-flow fractionation (AF4) with large-volume sample stacking and capillary electrophoresis (LVSS/CE), to attain EVs with high purity for downstream protein profiling. This hyphenated system first separates the EVs from the contamination of smaller serum proteins by AF4, and second resolves the EVs from the coeluted, nonvesicular matrix components by CE following LVSS. The optimal LVSS condition permits the injection of 10-fold more EVs into CE compared to the nonstacking condition without compromising separation resolution. Collection and downstream analysis of the highly pure EVs after CE separation were demonstrated in the present work. The high EV purity yields a much-improved labeling efficiency when detected by fluorescent antibodies compared to those collected from the one-dimension separation of AF4, and permits the identification of more EV-specific cargos by LC–MS/MS compared to those isolated by ultracentrifugation (UC), the exoEasy Maxi Kit, and AF4. Our results strongly support that AF4-LVSS/CE can improve EV isolation and cargo analysis, opening up new opportunities for understanding EV functions and their applications in the biomedical fields