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Mass spectrometry (MS)-based technology has evolved as a powerful tool for the analysis of a wide array of biomolecules. However, its application to complex peptidomics and proteomics samples, for example neuropeptides, has proven to be challenging due to their extreme chemical complexities and low abundances in biological tissue samples. This PhD dissertation is focused on addressing some of these challenges by developing novel micro-separation tools and coupling them with MS to achieve enhanced MS sensitivity and increased peptide and protein coverage with lower sample consumption and reduced experimental time. Both chromatography-based and electrophoresis-based separations as well as their couplings to MS have been explored in this work, including monolithic material-based chromatography, improved capillary electrophoresis (CE) separation and novel coupling of separations to mass spectrometric imaging. N-acryloxysuccinimide-based monolithic column has been fabricated with specific antibodies immobilized onto the column to enable the extraction of targeted neuropeptide family from biological matrices. Alternatively, enzymes were immobilized onto the column for highly efficient on-column enzymatic digestion for proteomics studies. A methacrylate-based monolithic column with C12 functional groups was developed and coupled to both electrospray ionization (ESI) MS and matrix-assisted laser desorption/ionization (MALDI) MS for reversed-phase liquid chromatography (RPLC). For capillary electrophoresis, two improved capillary isoelectric focusing (CIEF) systems have been developed, including membrane-assisted CIEF and immobilized pH-gradient CIEF to obtain reduced sample loss and enhanced MS signal for neuropeptide analysis. An improved sheathless pressure-assisted CE (PACE) system was also developed for coupling to MALDI MS. For novel separation-MS imaging coupling, three generations of interfaces have been designed to couple CE, LC and multi-dimensional separations to MS imaging (MSI) for quantitative analysis of complex peptides. Collectively, this work results in development of the new generation of micro-separation and sample preparation tools and their coupling to various mass spectrometric ionization and detection modes for sensitive and rapid analysis of complex peptides and proteins. The technological innovations have significantly expanded the toolbox for peptidomics/proteomics analysis and will enable more in-depth investigations on peptide function and regulation.
Edited by two of the pioneers of microcolumn chromatography and written by recognized experts in the field, this book summarizes advances in microcolumn liquid chromatography, capillary supercritical fluid chromatography and microelectrophoresis. Its unique combination of expert knowledge from leading laboratories in the USA, Japan and Switzerland, results in a particularly in-depth and comprehensive coverage of the various aspects of microcolumn separation methods.
This book provides a detailed description of various multidimensional chromatographic separation techniques. The editor first provides an introduction to the area and then dives right into the various complex separation techniques. While still not used routinely comprehensive chromatography techniques will help acquaint the readers with the fundamentals and possible benefits of multi-dimensional separations coupled with mass spectrometry. The topics include a wide range of material that will appease all interested in either entering the field of multidimensional chromatography and those looking to gain a better understanding of the topic.
This book provides a detailed description of technical elements of a microbore column liquid chromatograph suitable for use in trace analysis. It presents examples of analyses, especially from the spheres of biochemistry, pharmacology, and environmental analytical chemistry.
The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. More than 260 volumes have been published (all of them still in print) and much of the material is relevant even today--truly an essential publication for researchers in all fields of life sciences. Key Features * Liquid chromatography * Electrophoresis * Mass spectrometry.
Widely employed for separating and detecting chemicals in solution, separation techniques are most often applied in tandem, subsequently referred to as hyphenated methods. Hyphenated and Alternative Methods of Detection in Chromatography details the development and application of mass spectral detection techniques coupled with gas phase and liquid phase chromatographies. With contributions from experts in a variety of fields, as evidenced by the range of topics, the book describes the advantages and disadvantages of various separation techniques and addresses methods for cutting-edge applications such as proteomics research. Specifically, this book covers: The development of the LC-NMR and its application in chromatographic science, particularly in relation to the analysis of natural and environmental samples and in the study of reaction monitoring, biosynthetic analysis, and structural elucidation The application of LC-ICP in biological samples for the speciation of organoarsenic and organoselenium compounds; metal complexes in microorganisms, plants, and foods of plant origin; human body fluids and tissues; and in the detection of chemical warfare agents Electrochemical detection in liquid chromatography, system optimization, and applications in the analysis of flavonoids The use of chemiluminescence for microcolumn and capillary methods of separation The process of employing multidimensional separation methods as a means of identifying components in complex samples A detailed reference for those starting out in the field, this book also suits those who are more experienced yet require assistance in new directions. It is an ideal reference for senior undergraduate and postgraduate students wishing to supplement their learning experience. Containing over 800 references, this book is an excellent source of information within the field of hyphenated methods of chromatographic analysis.
The relatively new technique of solid phase microextraction (SPME) is an important tool to prepare samples both in the lab and on-site. SPME is a "green" technology because it eliminates organic solvents from analytical laboratory and can be used in environmental, food and fragrance, and forensic and drug analysis. This handbook offers a thorough background of the theory and practical implementation of SPME. SPME protocols are presented outlining each stage of the method and providing useful tips and potential pitfalls. In addition, devices and fiber coatings, automated SPME systems, SPME method development, and In Vivo applications are discussed. This handbook is essential for its discussion of the latest SPME developments as well as its in depth information on the history, theory, and practical application of the method. - Practical application of Solid Phase Microextraction methods including detailed steps - Provides history of extraction methods to better understand the process - Suitable for all levels, from beginning student to experienced practitioner