Applications of mass spectrometry in clinical chemistry and biomedical research.

摘要

Clinical chemistry is a medical discipline whose aim is to diagnose and assess disease by analysis of biological specimens. Modern laboratories can perform several hundred different tests using many different methods developed over the last century. The classical, more traditional assays are typically labour-intensive, not multiplexed (only measure one analyte or disorder per assay), expensive, require a long turnaround time, and may not provide adequate sensitivity and specificity. Developments in mass spectrometry (MS) and related technologies over the last two decades have provided solutions for many if not all of these shortcomings. While MS based applications have not yet been widely implemented in clinical chemistry laboratories, current developments will encourage the replacement of traditional methods as well as the expansion of clinically diagnostic endpoints. Indeed, modern MS can be used to simultaneously analyze and quantitate multiple biomarkers in a single analysis. Currently, no other technique exists that can provide a comparable multiplexed analysis. In this thesis, current MS and related technologies were developed and applied to several important but distinct clinical chemistry applications. In particular, novel and useful methodologies were developed and implemented for the analysis and quantification of small molecule and peptide drugs (antiretroviral drugs and calcitonin), disease markers (C-reactive protein) and the biochemical state of an important drug target (human aromatase). The aim of this thesis is to describe, illustrate and discuss several novel examples of MS based clinical chemistry methods and applications.;A novel capillary LC TOF-MS method for the determination of a peptide drug (salmon calcitonin), a thirty-two residue peptide hormone, in human plasma and urine was developed. The method requires only a few microliters of sample and is sufficiently robust to be implemented in a clinical chemistry setting. The assay provides unparalleled specificity, a useful alternative to immunoassays, and can be readily adapted to the multiplexed analysis of other peptide drugs.;A generally applicable procedure for the multiplexed quantitation of target proteins in complex mixtures is described. The method involves trypsinization in the presence of 13C6-labelled analog peptide internal standards i.e. Isotope dilution method. As an example of its utility, the method was used to quantitate rat urinary C-reactive protein (CRP) in a model of drug induced nephrotoxicity.;Finally, the application of MS and related technologies for identifying protein posttranslational modifications is discussed. A model membrane protein, human estrogen synthase, was thoroughly analysed for the presence of modifications. Extensive sequence coverage (90.2%) was obtained, a novel amino acid substitution was identified and the enzyme was confirmed to harbour an N-linked glycosylation site.;The development and application of a high throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the multiplexed therapeutic drug monitoring of eleven antiretroviral drugs in human plasma is described. The method was validated, tested against external quality control samples, and used for the therapeutic drug monitoring of more than 450 patient samples spanning nine months of analyses.