Chromatographic Analysis of Drug-Protein Interactions During Diabetes and Characterization of Human Serum Albumin Through Multidimensional Mass Spectrometry

Diabetes is a metabolic disease that can lead to the non-enzymatic glycation of serum proteins such as human serum albumin (HSA). Previous studies have indicated that glycation can affect the structure and function of these proteins. This dissertation describes the development of tools and techniques based on high performance affinity chromatography (HPAC) and multidimensional mass spectrometry to analyze the effects of glycation on the function and structure of HSA. A major portion of this research involved the utilization of HPAC to examine the effect of glycation on the binding of three second-generation sulfonylurea drugs and one third-generation sulfonylurea drug. These studies were conducted with HSA containing various levels of glycation. Frontal analysis and zonal elution competition studies were used to profile the binding properties of the drugs at the major and minor binding sites on samples of normal HSA and glycated HSA. Various trends in the binding affinity were observed for these drugs at the levels of glycation that were examined. A second portion of this research involved the development of an on-line immunoextraction format in HPAC for examination of drug-protein interactions with normal and glycated HSA. This study utilized a polyclonal anti-HSA antibody HPAC column to extract and bind normal HSA or glycated HSA. The adsorbed HSA or glycated HSA columns were then tested and used in a number of chromatographic formats to examine drug-protein interactions. Finally, a third portion of this research involved the use of multidimensional mass spectrometry to qualitatively profile the structure of HSA through sequence analysis. This work obtained sequence analysis results that were comparable to those found in a previous method involving matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In addition, collision-induced dissociation was used to confirm the identity of several peptide sequences that could be used as internal calibrants for future work involving glycated HSA. Advisor: David S. Hag

Affinity Chromatography: A Historical Perspective

Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generation of new binding agents, supports, and immobilization methods for this method are considered. Various applications of affinity chromatography are also summarized, as well as the influence this field has played in the creation of other affinity-based separation or analysis methods

Chromatographic immunoassays: strategies and recent developments in the analysis of drugs and biological agents

A chromatographic immunoassay is a technique in which an antibody or antibodyrelated agent is used as part of a chromatographic system for the isolation or measurement of a specific target. Various binding agents, detection methods, supports and assay formats have been developed for this group of methods, and applications have been reported that range from drugs, hormones and herbicides to peptides, proteins and bacteria. This review discusses the general principles and applications of chromatographic immunoassays, with an emphasis being given to methods and formats that have been developed for the analysis of drugs and biological agents. The relative advantages or limitations of each format are discussed. Recent developments and research in this field, as well as possible future directions, are also considered

Optimizing Sequence Coverage for a Moderate Mass Protein in Nano-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry

Sample pretreatment was optimized to obtain high sequence coverage for human serum albumin (HSA, 66.5 kDa) when using nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nESI-Q-TOF-MS). Use of the final method with trypsin, Lys-C and Glu-C digests gave a combined coverage of 98.8%. The addition of peptide fractionation resulted in 99.7% coverage. These results were comparable to those obtained previously with matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The sample pretreatment/nESI-Q-TOF-MS method was also used with collision-induced dissociation to analyze HSA digests and to identify peptides that could be employed as internal mass calibrants in future studies of modifications to HSA

Optimizing Sequence Coverage for a Moderate Mass Protein in Nano-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry

Sample pretreatment was optimized to obtain high sequence coverage for human serum albumin (HSA, 66.5 kDa) when using nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nESI-Q-TOF-MS). Use of the final method with trypsin, Lys-C and Glu-C digests gave a combined coverage of 98.8%. The addition of peptide fractionation resulted in 99.7% coverage. These results were comparable to those obtained previously with matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The sample pretreatment/nESI-Q-TOF-MS method was also used with collision-induced dissociation to analyze HSA digests and to identify peptides that could be employed as internal mass calibrants in future studies of modifications to HSA

Beneficial Effects of Insulin on Glycemic Control and β-Cell Function in Newly Diagnosed Type 2 Diabetes With Severe Hyperglycemia After Short-Term Intensive Insulin Therapy

OBJECTIVE—To evaluate whether treatment with insulin is advantageous compared with oral antidiabetes agents in newly diagnosed type 2 diabetes with severe hyperglycemia after short-term intensive insulin therapy

Low-Volume High-Intensity Interval Training in a Gym Setting Improves Cardio-Metabolic and Psychological Health.

BACKGROUND: Within a controlled laboratory environment, high-intensity interval training (HIT) elicits similar cardiovascular and metabolic benefits as traditional moderate-intensity continuous training (MICT). It is currently unclear how HIT can be applied effectively in a real-world environment. PURPOSE: To investigate the hypothesis that 10 weeks of HIT, performed in an instructor-led, group-based gym setting, elicits improvements in aerobic capacity (VO2max), cardio-metabolic risk and psychological health which are comparable to MICT. METHODS: Ninety physically inactive volunteers (42±11 y, 27.7±4.8 kg.m-2) were randomly assigned to HIT or MICT group exercise classes. HIT consisted of repeated sprints (15-60 seconds, >90% HRmax) interspersed with periods of recovery cycling (≤25 min.session-1, 3 sessions.week-1). MICT participants performed continuous cycling (~70% HRmax, 30-45 min.session-1, 5 sessions.week-1). VO2max, markers of cardio-metabolic risk, and psychological health were assessed pre and post-intervention. RESULTS: Mean weekly training time was 55±10 (HIT) and 128±44 min (MICT) (p<0.05), with greater adherence to HIT (83±14% vs. 61±15% prescribed sessions attended, respectively; p<0.05). HIT improved VO2max, insulin sensitivity, reduced abdominal fat mass, and induced favourable changes in blood lipids (p<0.05). HIT also induced beneficial effects on health perceptions, positive and negative affect, and subjective vitality (p<0.05). No difference between HIT and MICT was seen for any of these variables. CONCLUSIONS: HIT performed in a real-world gym setting improves cardio-metabolic risk factors and psychological health in physically inactive adults. With a reduced time commitment and greater adherence than MICT, HIT offers a viable and effective exercise strategy to target the growing incidence of metabolic disease and psychological ill-being associated with physical inactivity

Protective Unfolded Protein Response in Human Pancreatic Beta Cells Transplanted into Mice

Background: There is great interest about the possible contribution of ER stress to the apoptosis of pancreatic beta cells in the diabetic state and with islet transplantation. Methods and Findings: Expression of genes involved in ER stress were examined in beta cell enriched tissue obtained with laser capture microdissection (LCM) from frozen sections of pancreases obtained from non-diabetic subjects at surgery and from human islets transplanted into ICR-SCID mice for 4 wk. Because mice have higher glucose levels than humans, the transplanted beta cells were exposed to mild hyperglycemia and the abnormal environment of the transplant site. RNA was extracted from the LCM specimens, amplified and then subjected to microarray analysis. The transplanted beta cells showed an unfolded protein response (UPR). There was activation of many genes of the IRE-1 pathway that provide protection against the deleterious effects of ER stress, increased expression of ER chaperones and ERAD (ER-associated protein degradation) proteins. The other two arms of ER stress, PERK and ATF-6, had many down regulated genes. Downregulation of EIF2A could protect by inhibiting protein synthesis. Two genes known to contribute to apoptosis, CHOP and JNK, were downregulated. Conclusions: Human beta cells in a transplant site had UPR changes in gene expression that protect against the proapoptotic effects of unfolded proteins