Capillary electrophoresis coupled to mass spectrometry for clinical diagnostic purposes

Abstract

Capillary electrophoresis coupled to mass spectrometry for clinical diagnostic purposesThe introduction of fast, sensitive, and robust techniques for proteomic analysis into clinical practice represents a major step toward a new diagnostic approach of body fluids. In addition, proteomics emerges as a key technology for the discovery of disease biomarkers in various body fluids. However, even in relatively protein-deprived body fluids such as urine, the complexity and wide dynamic range of protein expression pose a considerable challenge to both separation and identification technologies. In the present review we discuss from a clinical point-of-view recent advances of the use of proteomics in clinical diagnosis as well as therapy evaluation. We focus on capillary electrophoresis coupled to mass spectrometry (CE-MS) and discuss CE-MS from an application point of view evaluating its merits and vices with regard to biomarker discovery. This review further presents examples of clinical applications of CE-MS for detection and identification of biomarkers in urine. IntroductionIt is well appreciated for centuries that body fluids contain information on the "internal" environment, i.e., the function of tissues and organs, which can be used as a gauge for the well being of the organism. Although this concept was created by Hippocrates more than 2000 years ago, standardized clinical laboratory tests were not developed before the 20th century. Moreover, a systematic discovery of biomarkers was made possible only with the discovery of antibodies and their use in immunological assays such as radioimmuno assay (RIA), ELISA, etc. Their increasing use enabled detection and quantification of distinct molecules -mainly peptides and proteins -in various body fluids. However, these techniques were limited by their requirement of prior knowledge of the molecule and a specific probe. New functional proteomic approaches appeared to circumvent these limitations. Spurred by advances in MS it was hoped that proteomics would allow the analysis of thousands of peptides and proteins in one sample [1][2][3][4][5][6], but several obstacles turned up soon. The high complexity and wide dynamic range of peptides in body fluids required pre-MS separation techniques that offer high resolution and could cope with a wide dynamic range of peptide concentrations [3]. Further, suitable software for interpretation of a vast number of generated MS spectra as well as comparison of the data was a major challenge [7].The approach towards solving most of these problems was to reduce data complexity, for example by arbitrarily collecting data on only a few polypeptides and also to reduce resolution of the analysis instrument. Both were accomplished with the surface-enhanced laser desorption/ionization (SELDI) technology -a complex solution of analytes was allowed to interact with an active surface (ion-exchange, hydro-

Urine in Clinical Proteomics

“…In addition, definition of disease-specific biomarkers in urine, and most likely in other compartments, is complicated by significant changes in the proteome during the day. These changes are likely caused by variations in the diet, metabolic or catabolic processes, circadian rhythms, and exercise as well as circulatory levels of various hormones (15). The reproducibility of any analysis is reduced by these physiological changes even if the analytical method shows high reproducibility.…”

Advances in Urinary Proteome Analysis and Biomarker Discovery

“…(66). As a consequence, the reproducibility of the assay is reduced as a result of these physiologic changes, even if the analytical method shows high reproducibility.…”

Urinary Proteomics and Renal Transplantation

“…(Solubilization of these low molecular weight proteins and peptides is a process with a major influence on the proteomics analysis) -> 30 KDa compounds can be analyzed in a mass spectrometer without additional manipulation -urinary protein is relatively stable probably due to the fact that urine "stagnates" for hours in the bladder -can be stored for several years at -80 °C without significant alteration of its proteome -not only the changes in the kidney and genitourinary tract are reflected by changes in the urinary proteome but also changes at more distant sites -it widely varies in protein and peptide concentrations mostly because of differences in the daily intake of fluid -standardization based on creatinine or peptides generally present in urine -definition of disease-specific biomarkers in urine, is complicated by significant changes in the proteome during the day -changes are likely caused by variations in the diet, metabolic or catabolic processes, circadian rhythms, and exercise as well as circulatory levels of various hormones Table 1. Advantages and disadvantages of urine as source of biomarkers (Decramer et al 2008, Fliser et al 2005, Kolch et al 2005, Omenn et al 2005, Schiffer et al 2006, and Theodorescu et al 2006 www.intechopen.com…”