Evaluation of Laboratory Diagnostic Methods

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Goals of this project are to improve clinical laboratory methods for diagnosis of disease. Studies include analysis of clinical laboratory practices, analysis of the accuracy of laboratory tests, and development of new tests and testing technologies. The major effort over the past year has been to analyze complex patterns of peptides and proteins in biological fluids by matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry. This technique allows simultaneous detection of more than 100 peptide and protein components in biological fluids. The primary challenge in applying MALDI TOF mass spectrometry to the analysis of biological fluids is in sample preparation. Biological fluids contain excess salt and a high abundance of several proteins such as albumin and immunoglobulins that interfere with the ability to detect less abundant components. Our efforts have been directed primarily at analysis of peptide and small protein components, which is the mass region yielding highest sensitivity and resolution by MALDI TOF mass spectrometry. Issues of optimal sample collection for analysis of plasma components have been examined. Solid phase extraction of urine was identified as a simple procedure for preparation of specimens for analysis of peptide and small protein components by MALDI TOF mass spectrometry. The relatively simple preparation of urine specimens and lack of overload by albumin may allow diagnostic evaluations of protein components for applications such as identification of kidney tubular defects and of overload proteinurias such as those occurring with myeloma. The potential for blood collection tubes to add extraneous materials that may interfere with mass spectrometric analyses was examined, and many types of tubes were found to add polymeric components that appear in mass spectra. A second major hurdles to the clinical application of MALDI TOF mass spectrometry has been the lack of procedures for internal standardization and for cross-comparison with existing quantitative methods for protein analysis in the clinical laboratory. We have compared the correlation of peak areas by mass spectrometry by investigators in the Department of Transfusion Medicine with quantitative immunoassay results performed in our laboratory, and this may serve as a useful approach for calibration and validation of quantitative measurements by mass spectrometry. Detailed analysis of small peptide components of lipoproteins has been performed. Analysis of purified high-density lipoprotein (HDL) shows the presence of more than 100 peptide components in the mass range of 1,000-5,000 daltons. Sequence analysis of peptides indicates that most of them are peptide fragments of abundant proteins that generally are not considered to be associated with lipoproteins. Many proteins in biological fluids undergo post-translational modifications. Our laboratory has studied optimal procedures for detecting selected post-translational modifications such as sulfation of tyrosine residues.