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

The Impact of Depression on Patient Outcomes in Hip Arthroscopic Surgery.

Background: Mental health impairments have been shown to negatively affect preoperative self-reported function in patients with various musculoskeletal disorders, including those with femoroacetabular impingement. Hypothesis: Those with symptoms of depression will have lower self-reported function, more pain, and less satisfaction on initial assessment and at 2-year follow-up than those without symptoms of depression. Study Design: Cohort study; Level of evidence, 3. Methods: Patients who were enrolled in a multicenter hip arthroscopic surgery registry and had 2-year outcome data available were included in the study. Patients completed the 12-item International Hip Outcome Tool (iHOT-12), visual analog scale (VAS) for pain, and 12-item Short-Form Health Survey (SF-12) when consenting for surgery. At 2-year follow-up, patients were emailed the iHOT, the VAS, and a rating scale of surgical satisfaction. Initial SF-12 mental component summary (MCS) scores Results: A total of 781 patients achieved the approximate 2-year milestone (mean follow-up, 735 ± 68 days), with 651 (83%) having 2-year outcome data available. There were 434 (67%) female and 217 (33%) male patients, with a mean age of 35.8 ± 13.0 years and a mean body mass index of 25.4 ± 8.8 kg/m Conclusion: A large number of patients who underwent hip arthroscopic surgery presented with symptoms of depression, which negatively affected self-reported function, pain levels, and satisfaction on initial assessment and at 2-year follow-up. Surgeons who perform hip arthroscopic surgery may need to identify the symptoms of depression and be aware of the impact that depression can have on surgical outcomes

Profiling the iron, copper and zinc content in primary neuron and astrocyte cultures by rapid online quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry

Metals often determine the chemical reactivity of the proteins to which they are bound. Each cell in the body tightly maintains a unique metalloproteomic profile, mostly dependent on function. This paper describes an analytical online flow injection quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method, which was applied to profiling the metal-binding proteins found in primary cultures of neurons and astrocytes. This method can be conducted using similar amounts of sample to those used for Western blotting (20-150 μg protein), and has a turnaround time of <15 minutes. Metalloprotein standards for Fe (as ferritin), Cu and Zn (as superoxide dismutase-1) were used to construct multi-point calibration curves for online quantification of metalloproteins by SEC-ICP-MS. Homogenates of primary neuron and astrocyte cultures were analysed by SEC-ICP-MS. Online quantification by external calibration with metalloprotein standards determined the mass of metal eluting from the column relative to time (as pg s-1). Total on-column Fe, Cu and Zn detection limits ranged from 0.825 ± 0.005 ng to 13.6 ± 0.7 pg. Neurons and astrocytes exhibited distinct metalloprotein profiles, featuring both ubiquitous and unique metalloprotein species. Separation and detection by SEC-ICP-MS allows appraisal of these metalloproteins in their native state, and online quantification was achieved using this relatively simple external calibration process. © 2013 The Royal Society of Chemistry

Pharmaceutical And Biomedical Applications Of Affinity Chromatography: Recent Trends And Developments

Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered

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